Introduction to High Performance Liquid Chromatography by cH4YUtp

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									Introduction to High Performance Liquid
Chromatography
In This Section, We Will Discuss:

 The differences between High Performance Liquid
  Chromatography and Gas Chromatography.

 The components of the high performance liquid
  chromatograph (HPLC).

 The separation process.

 The chromatogram.

 The most common modes of HPLC.




                                                    2
You’ve Got a Problem to Solve


                 I need a quantitative
                 separation of                         I’ll get
                 carbohydrates in some                 on it!
                 of our products
                 as soon as possible.



                              I’ll need a separation
                              technique.




                                                                  3
Separation Techniques




         I have two separation techniques in my lab,
         High Performance Liquid Chromatography
         and Gas Chromatography. Which should I use?
                                                       4
 Comparison of HPLC and GC

Sample Volatility                            Sample Polarity

HPLC                                           HPLC
• No volatility requirement                  • Separates both polar and
                                               non polar compounds
• Sample must be soluble
  in mobile phase                            • PAH - inorganic ions




                   GC                                        GC
                                                             • Samples are nonpolar
                 • Sample must be volatile
                                                               and polar




                                                                                      5
Comparison of HPLC and GC




                            6
Comparison of HPLC and GC

Sample Thermal Lability                   Sample Molecular Weight

HPLC                                         HPLC
• Analysis can take place
                                           • No theoretical upper limit
  at or below room
  temperature
                                           • In practicality, solubility is
                                             limit.



                 GC                                           GC
                • Sample must be able
                  to survive high                               • Typically < 500 amu
                  temperature injection
                  port and column




                                                                                        7
Comparison of HPLC and GC

Sample Preparation                            Sample Size

HPLC                                            HPLC
 • Sample must be filtered
                                              • Sample size based upon
                                                column i.d.
 • Sample should be in
   same solvent as mobile
   phase



                  GC                                         GC

                 • Solvent must be volatile                  • Typically 1 - 5 L
                   and generally lower
                   boiling than analytes




                                                                                    8
Comparison of HPLC and GC

Separation Mechanism                  Detectors

HPLC                                    HPLC
• Both stationary phase               • Most common UV-Vis
  and mobile phase take               • Wide range of non-
  part                                  destructive detectors
                                      • 3-dimensional detectors
                                      • Sensitivity to fg (detector
                                        dependent)

                GC                                      GC
                                                         • Most common FID,
               •Mobile phase is a                          universal to organic
                sample carrier only                        compounds




                                                                                  9
How can We Analyze the Sample?



    Carbohydrates
    1.   fructose
    2.   Glucose
    3.   Saccharose
    4.   Palatinose
    5.   Trehalulose
    6.   isomaltose                                               5




                                               2
                                                          3
  Zorbax NH2 (4.6 x 250 mm)          mAU
                                                              4
  70/30 Acetonitrile/Water                 1
                                                                      6
  1 mL/min Detect=Refractive Index

                                                   time


                                                                          10
Separations
                                    Separation in based upon differential
                         Injector
                                    migration between the stationary and
                                    mobile phases.
               Mixer                Stationary Phase - the phase which
                                    remains fixed in the column, e.g. C18,
                                    Silica

       Pumps                        Mobile Phase - carries the sample
                                    through the stationary phase as it
                                    moves through the column.
                           Column




                                            Detector




                                           Waste
    Solvents


           High Performance Liquid Chromatograph
                                                                            11
Separations
                         Injector                Chromatogram




               Mixer                mAU




       Pumps


                                          Start Injection   time

                           Column




                                              Detector




    Solvents


           High Performance Liquid Chromatograph
                                                                   12
Separations
                       Injector                Chromatogram




               Mixer              mAU




       Pumps


                                        Start Injection   time

                         Column




                                            Detector




    Solvents




                                                                 13
Separations
                       Injector                Chromatogram




               Mixer              mAU




       Pumps


                                        Start Injection   time

                         Column




                                            Detector




    Solvents




                                                                 14
Separations
                       Injector                Chromatogram




               Mixer              mAU




       Pumps


                                        Start Injection   time

                         Column




                                            Detector




    Solvents




                                                                 15
Separations
                       Injector                Chromatogram




               Mixer              mAU




       Pumps


                                        Start Injection   time

                         Column




                                            Detector




    Solvents




                                                                 16
Separations
                       Injector                Chromatogram




               Mixer              mAU




       Pumps


                                        Start Injection   time

                         Column




                                            Detector




    Solvents




                                                                 17
Separations
                       Injector                Chromatogram




               Mixer              mAU




       Pumps


                                        Start Injection   time

                         Column




                                            Detector




    Solvents




                                                                 18
Separations
                       Injector                Chromatogram




               Mixer              mAU




       Pumps


                                        Start Injection   time

                         Column




                                            Detector




    Solvents




                                                                 19
Separations
                       Injector                Chromatogram




               Mixer              mAU




       Pumps


                                        Start Injection   time


                       Column




                                            Detector




    Solvents




                                                                 20
Separations
                       Injector                Chromatogram




               Mixer              mAU




       Pumps


                                        Start Injection   time


                       Column




                                            Detector




    Solvents




                                                                 21
Separations
                       Injector                Chromatogram




               Mixer              mAU




       Pumps


                                        Start Injection   time


                       Column




                                            Detector




    Solvents




                                                                 22
Separations
                       Injector                Chromatogram




               Mixer              mAU




       Pumps


                                        Start Injection   time


                       Column




                                            Detector




    Solvents




                                                                 23
Separations
                       Injector                Chromatogram




               Mixer              mAU




       Pumps


                                        Start Injection   time


                       Column




                                            Detector




    Solvents




                                                                 24
Separations
                       Injector                Chromatogram




               Mixer              mAU




       Pumps


                                        Start Injection   time


                       Column




                                            Detector




    Solvents




                                                                 25
Separations
                       Injector                Chromatogram




               Mixer              mAU




       Pumps


                                        Start Injection   time


                       Column




                                            Detector




    Solvents




                                                                 26
Separations
                       Injector                Chromatogram




               Mixer              mAU




       Pumps


                                        Start Injection   time


                       Column




                                            Detector




    Solvents




                                                                 27
  The Chromatogram


to - elution time of unretained peak
tR- retention time - determines sample identity
                                        tR



                        tR
      mAU                                         Area or height is proportional
                                                  to the quantity of analyte.

                to


              Injection                 time

                                                                        28
HPLC Analysis Parameters



                      Mobile Phases



                      Flow Rate
                      Composition

                      Injection Volume
                      Column
                      Oven Temperature
                      Wavelength
                      Time Constant
                                         29
Modes of High Performance Liquid
Chromatography



   Types of Compounds      Mode        Stationary        Mobile Phase
                                       Phase
   Neutrals                Reversed    C18, C8, C4       Water/Organic
   Weak Acids              Phase       cyano, amino      Modifiers
   Weak Bases

   Ionics, Bases, Acids    Ion         C-18, C-8         Water/Organic
                           Pair                          Ion-Pair Reagent

   Compounds not           Normal      Silica, Amino,    Organics
   soluble in water        Phase       Cyano, Diol

   Ionics Inorganic Ions   Ion         Anion or Cation   Aqueous/Buffer
                           Exchange    Exchange          Counter Ion
                                       Resin
   High Molecular Weight   Size        Polystyrene       Gel Filtration-
   Compounds               Exclusion   Silica            Aqueous
   Polymers                                              Gel Permeation-
                                                         Organic




                                                                            30
HPLC Applications

                                                    Bioscience
        Chemical                                    proteins
                                                    peptides
        polystyrenes                                nucleotides
        dyes
        phthalates



                                  tetracyclines
                  Pharmaceuticals corticosteroids
                                  antidepressants          Consumer Products
                                  barbiturates
                                                                lipids
                                                                antioxidants
                                                                sugars
        Environmental
       polyaromatic hydrocarbons
                                                     Clinical
       Inorganic ions                                amino acids
       herbicides                                    vitamins
                                                     homocysteine

                                                                               31

								
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