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Chromatography Techniques

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					      Chromatography Techniques
Advances & Advantages in Analytical Chemistry



                                Mr.Rambabu
                            Executive -Application Support
                            Spinco Biotech Pvt. Ltd.
                                  Hyderabad
            Chromatography Techniques

o Concept of Chromatography



o Various Types of Chromatography
  Techniques
    o Principle & Instrumentation)
        o HPLC, UFLC, LCMS, GC &
           GCMS


o Applications




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            Concept of Chromatography
• Chromatography is an analytical method that the
  compounds are physically separated prior to measurement

• The main purpose of chromatography is to separate and
  quantify the target sample in the matrix


                                        Liquid
                                    Chromatography


                                         Gas
    Chromatography
                                    Chromatography


                                    Supercritical-fluid
                                    Chromatography
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        History of chromatography
M. Tswett : first developer of chromatography


                                  Petroleum ether



                               Chlorophylls


       CaCO3




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        Separation Mechanism

Compounds are separated because the molecules
move at different rates in the column.




                                                1
                                                    2




             column



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                  Separation Mechanism
        Due to different interaction between stationary phase
        and different sample, the molecules move at different
        rate, therefore separation can be done.

Mobile Phase



                                                          1
                                                                2

                Stronger                  Weaker
               interaction              interaction

                       Stationary Phase


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                 Chromato. . . ???
・Chromatography
    Method for Separation
・Chromatograph
                                                        tR     : Retention time
   Instrument for Chromatography
                                                        A : Area

・Chromatogram                                           h : Height


   Data of Chromatography                               tR
                                                                    Peak



                                               Signal
. Chromatographer
                                                              h
     Analyst                                                         A
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             Why use HPLC?
• Simultaneous Analysis
• High Resolution
• High Sensitivity (ppm-ppb)
• Good repeatability
• Small sample size
• Moderate analysis condition
   - no need to vaporize the sample like GC
• Easy to fractionate the sample and purify
• No destructive for many detectors
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                         Scope of HPLC
       Field                           Typical mixtures
                    Antibiotics, sedatives, steroids, analgesics, crude
Pharmaceuticals
                    drugs, cosmetics
                    Amino acids, proteins, peptides, carbohydrates,
Biochemical
                    lipids, enzymes, medicines, hormone
                    Mycotoxins, additives, saccharides, amino acids,
Food products
                    vitamins, fatty acid, coloring agents, antibacterials
Industrial          Condensed aromatics, surfactants, propellants,
chemicals           dyes, polymers, plasticizers
Forensic chemistry Drugs, poisons, blood alcohol, narcotics
Environmental       Inorganic ions, organic acids, agricultural chemicals,
field               pesticides, herbicides, phenols,
                    Bile acids, drug metabolites, urine extracts,
Clinical medicine
                    estrogens
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        Separation Modes

•   Normal phase chromatography

•   Reversed phase chromatography

•   Ion chromatography

•   Size exclusion chromatography

•   Affinity chromatography


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           Scope of HPLC
                                                106


    Gel           Size               Gel
                                                105
 permeation     exclusion        filtration




                                                      Molecular weight
                                                104


                                                103
Normal           Reversed               Ion
 phase            phase              exchange
                                                102




                Nonionic polar

Nonpolar                             Ionic
   Water-insoluble          Water-soluble

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              Increasing polarity
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               Instrumentations

Modular HPLC
• Possible configurations
• Solvent delivery pumps
• Sample injectors
• Column ovens
• Detectors
Integrated HPLC
• LC-2010




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                       Isocratic System


                                         Column
                                                        Detector
                      Injector
            Pump                           Oven


Mobile Phase
                                                               Data
                                                            processor
  Simple system with one pump and one solvent reservoir.
  If more than one solvent is used, solvents should be premixed.


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            Low-pressure Gradient System


                                             Column         Detector
                         Injector
             Pump                              Oven
low pressure
gradient valve                                           Data
                                                       processor



                                 •One pump used to control 4 reservoirs;
                                 •Mixing is done before pump.
                                 •On-line degasser is necessary.
A     B          C   D

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           High-pressure Gradient System

A

    pump


             Mixer
B
                                          Column        Detector
    pump              Injector
                                           Oven
                                                     Data
                                                   processor
C


    pump    • Excellent gradient accuracy.
            • 2-3 pumps required - one pump per solvent used.
            • On-line degassing may not be critical.
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                      Flexibility
   Modular type provides excellent flexibility.




Isocratic system         Fully automated gradient system
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                  Outline of LC-2010
Reservior Tray

                                                System Controller
 Auto sampler


Column Oven
                                                UV detector
Degassing Unit

  Pump Unit

 Low pressure
gradient device   http://pharmacy2011foru.blo
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            Detectors for HPLC

• UV-VIS   Ultraviolet / Visible detector
• PDA      Photodiode Array detector
• RF       Fluorescence detector
• CDD      Conductivity detector
• RID      Refractive Index detector
• ECD      Electrochemical detector
• ELSD     Evaporative light scattering detector
• MS       Mass spectrometer detector




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   Ultraviolet / Visible Detector
Advantage:
• Sensitivity is high
• Relative robust to temperature and flow rate change
• Compatible with gradient elution


Disadvantage:
• Only compounds with UV or visible absorption could
  be detected.
Additional Functions
• Dual Wavelength mode
• Wavelength Time Program mode
• Wavelength Scan mode
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         Photodiode Array Detector

              Sample Cell           Grating



                                                  One element detects
D2 / W lamp                                       one absorbance at
                                                  one wavelength.




                   512 Elements Photodiode Array


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              Photodiode Array Detector
                      3-D Data
                                      Spectrum

Chromatogram
 Absorbance




                          Time
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                               PDA Detector
Advantages:
•   PDA Detector could analyze a sample simultaneously at many
    different wavelengths.
•   UV Visible spectra are useful for compound identification, checking
    peak purity, as well as finding the optimum absorbance for the
    compounds.
•   UV Visible spectra of many compounds could be stored in the
    spectrum libraries, which are useful for compound identification.
•   Relatively robust to temperature and flow rate fluctuations
•   Compatible with gradient elution.

Disadvantages:
•   Slightly less sensitive than UV-Visible detector.


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                Fluorescence of Compounds
Fluorescence is a type of luminescence in which the light
energy is released in the form of a photon in nanoseconds to
microseconds
                           Non-radiation transition
   S1
                                       Non-radiation transition
   T1

        Light absorption

                   Fluorescence
                                        Phosphorescence


   S0
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Fluorescence Detector




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                 Fluorescence Detector
Advantage
• Sensitivity is higher than UV-Vis detector
• Selectivity is high because relatively few compounds
  fluorescence
• Compatible with gradient elution

Disadvanage
• Difficult to predict fluorescence
• Greatly affected by environment
   – Solvent
   – pH
   – Temperature
   – Viscosity
   – Ionic strength
   – Dissolved gas
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             Refractive Index Detector (1)


Photodiode
                     Reference                      Refraction




                                                  W Lamp
                Sample



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               Refractive Index Detector

Advantage
Responds to nearly all solutes
Unaffected by flow rate


Disadvantage
Not as sensitive as most other types of detectors
Could not be used with gradient elution




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                  Refractive Index Detector
                            Application Example

• Analytical Conditions
   – Column : Shim-pack CLC-NH2
   – Mobile phase : Acetonitrile /
     water = 70/30
   – Flow rate : 1.0 mL/min
   – Temperature : Ambient
• Peaks
     1. Glycerol
     2. Xylose
     3. Fructose
     4. Glucose
     5. Sucrose
     6. Manose
     7. Lactose        http://pharmacy2011foru.blo
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                  SHIMADZU GROUP


                  130th


….the future of
    HPLC
             1.World’s first web control
               ◆ World’s first Web Control
                 Internet Explorer to access to the Prominence (no special software required)



                                                                                            Status summary


                                                                                            ・To show working
                                                                                             condition of the
                                                                                             systems in a
                                                                                             working group,
                                                                                             system check result,
 HPLC Explorer                                                                               and maintenance
                                                                                             information.
* Displays all
  HPLC systems
  on the network
* One workgroup
  includes max 10
  HPLC systems.                                                                                 Instrument monitor

                                                                                            ・To monitor
                                                                                             parameter setting
                                                                                             conditions and
                                                                                             baseline of chosen
                                                                                             system.




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          1.World’s first web control
◆ Access to the instruments via Ethernet
  --> CBM-20A/20Alite
      - No need to dedicate a client PC
        - Any PC on network can access to Prominence system.




                                                    PC-Bからprominence-Aにログイン
                                                    PC-Aからprominence-Aにログイン
                                                     Prominenceでの使用環境
                                                        prominence-Aからログアウト
                                                    PC-Aからprominence-A/Bをモニタ
                                                    PC-Bからprominence-A/Bをモニタ



                         従来の使用環境
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                         CBM-20A/20Alite Features --Network example--

Reserve
 room               AP                             Office
                                                                                      to backbone network


                                                                  Control/data acquisition of         Control/data acquisition of        Monitor/setting of prominence
                                                                 prominence B, C, D, E, and          prominence B, C, D, E, and                 B, C, D, E, and
                                                                communication with backbone         communication with backbone          communication with backbone
                                                                           network                             network                              network

                                                                         LCsolution                          LCsolution                           Client PC




  prominence E            LCsolution                      AP


 Control/Data acquisition of Prominence E



                                                           Workgroup 1                                                    Workgroup 2
 Lab




  prominence A            Empower            prominence B           CLASS-VP            prominence C            LCsolution          prominence D           LCsolution
                                              SPD-M20A
     Monitor/setting of prominence A
      Data acquisition by Empower
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                                            Control/data acquisition of prominence B Control/data acquisition of prominence C, D Control/data acquisition of prominence C, D

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     2.World’s only system with data buffering


◆ LC workstation connection via network
   HPLC system control and data processing by
   LCsolution/CLASS-VP on the network..

◆ World's only data buffering capability
   Even in case network problem occurs, precious
   analytical result is secured. (Max. one analysis data
   less than 24 hours at 500ms sampling.)

◆ Automation capability                                                     CBM-20A
   Auto-purge, auto-startup, auto-shutdown are
   possible for modular type HPLC systems for the
   first time.




                                                                         CBM-20Alite
                                                           to be installed inside solvent delivery unit or
                                                                            autosampler
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      3.World’s quietest HPLC pump
                                                                   Low pressure GE
                                                                        valve
World's best solvent delivery performance
   → LC-20AD/20AB

        ・ Flow rate resolution 3nL/step is
          achieved with micro stroke volume plunger

        ・ Ideal front-end for LC-MS for ultra trace
            high sensitivity analysis

       * Wide flow rate range from 0.0001 to
          10 ml/min
                                                         Inside view of LC-20AT




    LC-20AD                     LC-20AT               LC-20AB
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4.World’s Lowest Degassing volume
World's lowest degassing volume
DGU-20A3/20A5


    Low Capacity Degasser uses Teflon AF Film and Degassed Flow capacity
    is as low as 170 micro liter.

    Degassing volume allows a dramatic decrease in time required for
    replacing mobile phases and stabilization.


Specifications :

•   Number of Flow lines : DGU-20A3 – 3 lines, DGU-20A5 – 5 lines
•   Maximum operating Flow rate : 20 ml / min per flow line
•   Internal capacity : 380 ul / flow line
•   Tubing 3 mm OD x 1.5 mm id PTFE tubing




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       5. World’s fastest Auto Sampler

◆ World's fastest sample injection
   Fast needle drive technology developed for the LC-
   2010HT and SIL-HT is further improved, and achieved
   the world's fastest - 10 seconds for 10uL injection.


◆ ◆ In MTP/DWP setting, vials can be used.
   Control rack to accommodate 10 x 1.5mL vials is
   available.                                             SIL-20AC (right) and Rack Changer
                                                                         (left)
                                                              Sample Rack       Control Rack
◆ Rack Changer for large number of sample
  processing
   Switching max 12x MTP/DWP plates, continuous
   analysis is possible. * Standard model and Cooling
   model (Rack Changer C)




                              http://pharmacy2011foru.blo
                                       gspot.com/                   Inside of SIL-20AC
       6. World’s Cleanest Auto-Sampler
     ◆Near zero sample carry over
        Needle tip and seal shapes are modified. World's best low sample carry over
        performance is achieved. Using optional rinsing kit, multi-liquid rinse is possible.


                    SIL-HT                            SIL-20A
                                 Remained sample
                                                                             reduced to 1/4

                                                                       Minimized touch area


                      SIL-20A provides good result even                      With rinsing pump, sample carryover is
                      with adsorptive compound.                              not detected.
with HPLC from
   vendor A:
     0.08 %
                                     0.0007 %                                                 Not
                                                                                              detected




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                                Sample carryover test using Chlor-hexidine
         7. World’s most sensitive PDA detector

◆ World's highest sensitivity
   Noise level is reduced by reducing noise derived from
   electrics.
   <Noise>

   SPD-M20A: less than 0.6×10-5AU (high sensitivity mode)


◆ Improved stability
   Baseline is more stabilized with light source correction
   mechanism.                                                     SPD-M20A

   Temperature controlled cell as standard to minimize baseline
   change by room temperature change.



◆ D2 lamp/W lamp simultaneous lit mode




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   8. World’s most sensitive UV-VIS detector

◆ World's highest sensitivity
   Noise level is reduced by reducing noise derived from
   electrics.
   <Noise>

   SPD-20A/20AV: less than 0.25×10-5AU



◆ ◆ Improved stability

   Baseline is more stabilized with light source correction       SPD-20A/20AV
   mechanism.

   Temperature controlled cell as standard to minimize baseline
   change by room temperature change.




◆ Low Pressure Mercury Lamp is incorporated
   Easy wavelength calibration in UV region.



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                         SPD-20A/20AV/M20A Performance
                                           --Temperature controlled cell--
◆ Temperature controlled cell is equipped as standard so that
  influence of ambient temperature change to analysis is
  reduced.
         ・Stable baseline improves analysis reliability for trace component analysis.

            without cell temperature control
                       セル温調なし                                                     セル温調40℃
                                                                     cell temperature controlled at 40deg.C
mAU                                               ℃           mAU                                             ℃
                                                   40                                                         40


                 Cell temperature                                                    セル温度
                         セル温度                      35                           Cell temperature              35
           Room                                                        Room
           室温
         Temperature                                                  室温
                                                                    Temperature
15                                                 30         15                                              30


                                                   25                                                         25
10                                                            10

                       ベースライン
                       Baseline                    20                                                         20
5                                                              5
                                                                                   Baseline
                                                                                   ベースライン


0                                                              0

     0          30         60
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                                      90          120 min          0          30         60        90     120 min
                           Effect of temperature controlled cell --baseline change when ambient
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                                    temperature is changed from 20 deg.C to 30 deg.C.
        9. World’s best front-end HPLC for
                    LC-MS/MS
    ◆ Higher throughput
    •   World's fastest sample injection makes analysis cycle less than 1 minute possible.
    •   Ideal front-end for LC-MS for ultra trace high sensitivity analysis
    •   Rack changer processes upto 12 MTP/DWP for high throughput use.
    •   Data reliability improvement for ultra trace sample analysis
    •   Powerful support for LC-MS/LC-MSMS analyses




0              0.5           1             1.5            2            2.5             3 min
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10. World’s best LC Virtual advisor
                            • Shimadzu Lc Virtual
                              advisor

                                      • 24/7/365




                               • www.shimadzu.com/Lcva




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                                    Summary

•   World’s first web-based instrument control
•   World’s only system with Data Buffering
    capability
•   World’s quietest HPLC pump
•   World’s lowest degassing volume
•   World’s fastest Auto-sampler
•   World’s cleanest Auto-sampler
•   World’s highest sensitive PDA detector
•   World’s most sensitive UV-VIS detector
•   World’s best Front End HPLC for LC-MS/MS
•   World’s best LC Virtual advisor multimedia tool



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    eXtreme Resolution LC
   Prominence UFLCXR series
Analytical & Instruments Division
        LC Business Unit
    New drivers for customer to
          buy New HPLC
Driver     Productivity Improvement            Better quality of
                                               analysis
Why they   •Shorten development time           Can assure better
need       •Improve Cost/Analysis              quality of their Products.
this?
Who need Pharma Lead Discovery, High           •API provider etc.
this?    throughput screening                  •Who needs impurity
                                                  analysis
What is    •Fast injection cycle               •Hi-Resolution
needed     •High sample capacity               •Wide dynamic range
for HPLC   •MS compatibility( Low carry        •Low carry over
           over)
           •Longevity
Our        UFLC+XR-ODS                          UFLC XR+XR-ODSⅡ
Solution


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              High speed & High
                  resolution
High throughput

                  UFLC
             High throughput
              screening etc.
                                     UFLC XR
                                   Impurity analysis
                                for quality control etc.

              Conventional
                 HPLC




                                                           Resolution
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     Design concept of UFLC-XR
• No compromise in basic performance!
   – Waters gave up direct injection in UPLC! that result in poor
     reproducibility, less linearity and much carry over.
   – Agilent could not improve their fatal design that will not
     satisfy both High Speed and Low Carry over at the same
     time.

   – Thanks to our DIIMS technology (Direct Injection with
     Isolated Metering System), Only Shimadzu can achieve
     High Speed, Low Carry over, High precision and good
     linearity at the same time.




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       To realize extreme resolution
Prominence UFLC-XR series have

• Optimized flow line for less dispersion.
   – tubing,needle seal, Valve
• Increased pressure tolerance
   – up to 66MPa
• Higher resolution column
   – 2.2um core with 8nm pore
• Enhanced DIIMS for high performance in eXtream
  pressure.
   – Same repeatability and low carry over even in
     60MPa.

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 Outline of Prominence UFLC-XR series
• Maximum allowable pressure: 66MPa
• New Products for Prominence UFLCXR series
   – LC-20ADXR                :New
   – SIL-20AXR / SIL-20ACXR        :New
   – CBM-20A                  :F/W Ver.1.2
   – LCsolution               :Ver.1.23
   – Shim-pack XR-ODSII Column :New

• Upgrade KIT for current 20A products
  are available
       *for LC-20AD
       *for SIL-20A/C series
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   Solvent delivery pump LC-20ADXR
• What’s new in LC-20ADXR


   – Pressure Sensor     :70MPa scale pressure sensor
   – Drain Valve           :Modified Sealing performance
   – Inlet Check Valve    :Modified ball/seat design
   – Plunger Seal          :GFPUHMWPE
   – Seal wash KIT         :Standard
   – F/W version            :Ver. 1.21




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 Auto sampler SIL-20A/CXR
• What’s new in SIL-20A/CXR


   – Enhanced DIIMS
   – High pressure valve ASSY          : Reduced volume,
     higher pressure
   – Needle Seal              : Reduced volume, higher
     pressure
   – Sample loop              : Reduced volume
   – Outlet tube              : Improved handling
   – F/W                      : Ver. 1.20

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          Shim-pack XR-ODSⅡ
•
                           Column
    For higher resolution analysis
   – Raised Maximum pressure from 30MPa to 60MPa
   – Decreased pore size from 12nm to 8nm to realize high
     separation.
   – Added 150mm length for high separation use.
• Line up for Shim-pack XR-ODSⅡ

       ID           Length              Max. Press      Pore size
     2mm       75,100,                   60MPa            8nm
               150mm
     3mm       75,100,                   60MPa            8nm
               150mm
    Ref: XR-ODS column
         ID               Length           Max. Press     Pore size

        2mm          30, 50, 75,100mm       30MPa          12nm

        3mm          30, 50, 75,100mm       30MPa          12nm

       4.6mm         30, 50, 75,100mm       30MPa
                               http://pharmacy2011foru.blo 12nm
                                        gspot.com/
Performance of UFLC-XR series
• Fast and sharp.
  – Double the resolution with 1/4 of
    separation time.
    mAU

  150
                              1                        Shim-pack XR-ODSⅡ (3 mm i.d.  150 mm , 2.2 m)
                                                        Flow rate : 1.3 mL/min
                                      2
  125
                                            3               N for Peak 7 : 28,100
  100
                                                  4
   75                                                           5
                                                                                 6
   50
                                                                                                  7
   25

    0

        0.0   0.5                     1.0             1.5              2.0              2.5               min
        mAU
                                                                                                  3.0
  125                 1                                Shim-pack VP-ODS (4.6 mm i.d.  150 mm , 4.6 m)         Peaks; 1: Acetophenone
  100
                          2                             Flow rate : 1.0 mL/min                                         2: Propiophenone
                                  3                         N for Peak 7 : 14,800                                      3: Butyrophenone
   75                                       4                                                                          4: Valerophenone
                                                      5
   50
                                                                             6
                                                                                                                       5: Hexanophenone
                                                                                              7
   25                                                                                                                  6: Heptanophenone
    0
                                                                                                                       7: Octanophenone
        0.0     2.5                         5.0                 7.5              10.0                     min
                                                                                              12.5
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  Performance of UFLC-XR series
• extreme resolution
      mAU
100
                                    1

 75
                                                                            Shim-pack XR-ODSⅡ (3 mm i.d.  100 mm , 2.2 m)
 50                                          2                4
                                                  3
                                                                  5         6       8                                                               Chromatographic conditions;
                                                                                          9
 25                                                                             7
                                                                                              10          11
                                                                                                               12                13                 mobile phase
  0
                                                                                                                                                       A: water/THF (8/2,v/v)
      0.0   0.5   1.0             1.5       2.0         2.5           3.0           3.5       4.0   4.5             5.0   5.5          6.0    min      B: acetonitrile
100
      mAU                                                                                                                                           (Gradient Elution)
                              1


 75
                                                                                                                                                       1. Formaldehyde
                                                                                                                                                       2. Acetoaldehyde
                                                                                Shim-pack XR-ODS (3 mm i.d.  75 mm , 2.2 m)
                                                                                                                                                       3. Acetone
 50                                     2         3     4                                                                                              4. Acrolein
                                                              5                     8                                                                  5. Propionaldehyde
                                                                            6
 25
                                                                                7         9                                                            6. Crotonealdehyde
                                                                                               10     11         12             13                     7. 2-Butanone
                                                                                                                                                       8. Methacrolein
  0                                                                                                                                                    9. n-Butylaldehyde
      0.0               1.0                       2.0                                   3.0                4.0                       5.0     min       10. Benzaldehyde
                                                                                                                                                       11. Valeraldehyde
                                                                                                                                                       12. m-Tolualdehyde
                                                                      http://pharmacy2011foru.blo                                                      13. Hexaldehyde
                                                                               gspot.com/
UFLC Applications




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       Fast LC Application (1)
 ◆ PTC-amino acids
     → 6 times faster than conventional column (5m,4.6 mm i.d. 
     150 mm)
                mAU
                    2. than high speed column (3m, 4.6 mm i.d.  75
     → 2 times faster Glu
          250
            1. Asp           3. Ser              1. Formaldehyde
                                                          18
                                                 2. Acetoaldehyde
     mm)
            4. Gly  5. His   6.Arg
            7. Thr  8. Ala   9. Pro              3. Acetone
        200       10. Ammonia     11. Tyr      12. Val                                     4. Acrolein
                                                                                           5. Propionaldehyde
                  13. Met         14.Cys-Cys   15. Ile
                                                                             14            6. Crotonealdehyde
                  16. Leu         17. Phe      18. Lys                                     7. 2-Butanone
          150     (each 400 pmol/1L)                                 11 12             16 8. Methacrolein
                                                                 9                15
                               1                      5 6                   13             9.17
                                                                                              n-Butylaldehyde

                                               34           78                            10. Benzaldehyde
                                                                                          11. Valeraldehyde
          100                                                                             12. m-Tolualdehyde
                                   2                                                      13. Hexaldehyde
                                                                                          (each 120 nL/4L)

           50                                                    10


            0
            0.0          0.5           1.0      1.5          2.0      2.5         3.0             3.5           min


Chromatographic conditions; column: Shim-pack XR-ODS (2.2m, 3.0 mm i.d.  75
mm), mobile phase: 10 mMol/L (potassium)phosphate <pH 7.0> for A and acetonitrile
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for B (A/B: 95/5 for 0.3 min then 95/5 to 60/40 in 3.4 min), flow rate: 1.2 mL/min,
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temperature: 40 C, detection: absorbance at 254 nm.
           Fast LC Application (2)
◆        2,4-DNP-aldehydes/ketones
   → 6 times faster than conventional column (5m,4.6 mm
   i.d.  150 mm)
              mAU
   → 3 times faster than high speed column (3m, 4.6 mm
        100
                 1                       1. Formaldehyde

   i.d.  75 mm)
                                         2. Acetoaldehyde
                                         3. Acetone
                                         4. Acrolein
         75                                                                 5. Propionaldehyde
                                                                            6. Crotonealdehyde
                                                                            7. 2-Butanone
                                                                            8. Methacrolein
                                                                            9. n-Butylaldehyde
         50                  2        4                                    10. Benzaldehyde
                                  3
                                                                           11. Valeraldehyde
                                          5           8                    12. m-Tolualdehyde
                                              6                            13. Hexaldehyde
                                                  7       9                (each 120 nL/4L)
         25                                                   10   11 12
                                                                                   13


          0

           0.0         1.0        2.0                 3.0          4.0                  5.0      min


Chromatographic conditions; column: Shim-pack XR-ODS (2.2m, 3.0 mm i.d.  75
mm), mobile phase: water/THF (8/2,v/v) for A and acetonitrile for B (A/B: 8/2 to 5/5 in
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5 min), flow rate: 1.2 mL/min, temperature: 50 C, detection: absorbance at 360 nm.
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            2,4-DNP-Aldehydes/ Ketones UFLC-LCMS

7.5 (x10,000)                                                                                 (x10,000)
                      Shim-pack XR-ODS + APPI-MS                                        5.0                       Bland-A ODS (5 µm) + APPI-MS
7.0

6.5                        2     3               8   10    11         13
                       1                                                                4.0
                                                                12
6.0                              4 5             9
                                             7                                          3.0
5.5
                                        6
5.0                                                                                     2.0
4.5
                                                                                        1.0
4.0

3.5                                                                                     0.0
                                                                                              0.0         5.0      10.0   15.0    20.0     25.0         min
3.0
                                                                                                    (x10,000)
2.5                                                                                           5.0               Bland-A ODS (5 µm) + APCI-MS
2.0                                                                                           4.0
1.5
                                                                                              3.0
1.0
                                                                                              2.0
0.5
                                                                                              1.0
0.0
      0.0       1.0        2.0         3.0           4.0             5.0     min              0.0
Detector:                            LCMS-2010EV                               0.0   5.0   10.0 15.0                             20.0    25.0     min
Column:                              Shim-pack XR-ODS (50 mmL x 2.0 mmI.D. Particle size: 2.2 µm) or
                                     Bland-A ODS (150mmL x 2.0 mmI.D. Particle size: 5 µm)
Mobile phase:                        A; water/tetrahydrofuran = 80/20
                                     B; acetonitrile
Flow rate:                           0.5 mL/min (Shim-pack XR-ODS), 0.2 mL/min (Bland-A ODS)
Injection volume:                    2 µL (27pg each)
Time program:                        20%B (0 min) - 50%B (4.0 min) - 50%B (4.2 min)

APPI negative, SIM, m/z 209 (1: formaldehyde), m/z 223 (2: acetaldehyde), m/z 237 (3: acetone, 5: propionaldehyde),
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m/z 235 (4: acrolein), m/z 249 (6: crotonaldehyde, 8: methacrolein), m/z 251 (7: 2-butanone, 9: n-butyraldehyde),
m/z 285 (10: benzaldehyde), m/z 265 (11: valeraldehyde), m/z 299 (12: m-tolualdehyde), m/z 279 (13: hexaldehyde)
                                                                           gspot.com/
 LCMS spectrum of 2,4-DNP-Aldehydes and Ketones
       Inten. (x1,000,000)                                             Inten. (x1,000,000)                                             Inten. (x1,000,000)
                     467                                                                537                                     1.25              421
 1.5                                                      1     1.00                                                     2                                                                    3
                                                                                                                                1.00
                                                                0.75
 1.0                                                                                                                            0.75
                                                                0.50
                                                                                                                                0.50
 0.5
                               701                              0.25                                                            0.25      210                    632
                                                                          268                         807                                                                      843
        233
                                             935
 0.0                                                            0.00                                                            0.00
         250             500      750          1000       m/z            250          500           750           1000    m/z            250          500                750           1000    m/z
     Inten. (x1,000,000)                                             Inten. (x100,000)                                                 Inten. (x100,000)
1.00                  537                                                       407                                                                 451
                                                                 7.5
                                                          4                                                              5                                                                    6
                                                                                                                                 7.5
0.75
                                                                 5.0
0.50                                                                                                                             5.0
                                                                                                          815
          268                                                    2.5                          611                                                                  677           903
0.25                                                                      203                                                    2.5
                                                                                                                                          225
          207319
                                       806         1075
0.00                                                             0.0                                                             0.0
         250             500      750          1000       m/z            250          500           750           1000    m/z            250          500                750           1000    m/z
       Inten. (x100,000)                                               Inten. (x1,000,000)                                             Inten. (x100,000)
                                     763                        1.00                                747                          7.5                       557
1.00                                                      7                                                              8                                                                    9
                   381
                                                                0.75            373
0.75                                                                                                                             5.0
                                                                0.50
0.50
                                                                                                                                 2.5
0.25                                                            0.25
          215                                                                                                                            251 327
           281 347
                     441
0.00                                                            0.00                                                             0.0
         250             500      750          1000       m/z            250          500           750           1000    m/z            250          500                750           1000    m/z
       Inten. (x100,000)                                               Inten. (x1,000,000)                                             Inten. (x100,000)
                                     784.4                      1.25                                      834.6                                                                      972.5
 5.0                                                  10                                                                 11                                                                   12
                                                                1.00                                                             5.0

                                                                0.75
 2.5
                                                                0.50                                                             2.5

                                 740.6                          0.25                                                                                                              928.3
 0.0
                               702.5
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                                                                0.00                                                             0.0
                                                                                                                                                                                890.5

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         250             500      750          1000       m/z        250 500   750  1000 m/z                                             250          500                750           1000    m/z
           Fast LC Application (3)
◆ Tryptic digest of ovalbumin
   → High-speed separation by using the column with 19,500 in
  theoretical plate number
 → Peak width at half height is 1.2 sec
             The
          ( 40mAU average of the three peaks between 2.5 and 3.5
                                               1. Formaldehyde
                                               2. Acetoaldehyde

 minutes)
                                               3. Acetone
                                               4. Acrolein
                                                                      5. Propionaldehyde
                                                                      6. Crotonealdehyde
              30                                                      7. 2-Butanone
                                                                      8. Methacrolein
                                                                      9. n-Butylaldehyde
                                                                     10. Benzaldehyde
              20                                                     11. Valeraldehyde
                                                                     12. m-Tolualdehyde
                                                                     13. Hexaldehyde
                                                                     (each 120 nL/4L)
              10



               0
               0.0             2.5             5.0             7.5                  min


    Chromatographic conditions; column: Shim-pack XR-ODS (2.2m, 3.0 mm i.d.  100
    mm), mobile phase: 0.1 % TFA aqueous solution for A and 0.1 % TFA acetonitrile solution
                                 min), flow rate: 1.0 mL/min, temperature: 50 C, detection:
    for B (A/B: 9/1 to 5/5 in 10 http://pharmacy2011foru.blo
    absorbance at 214 nm, sample: 800 pmol/4L.
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                         Pigments analysis                               Column     Shim-pack XR-ODS
mA U
                                                                                    (3.0 mm i.d.×75 mm)
500                                                                       Mobile    A) 50mmol/L
          Peaks                                                           Phase      (Anmonium) Acetate
           1: Yello w 4        7: Green 3                                            Buffer (pH 4.7)
           2: Red 2            8: Blue 1                                            B) 50mmol/L
400                                                     8                            (Anmonium) Acetate
           3: Blue 2           9: Red 106                                            Buffer (pH 4.7) /
           4: Red 102         10: Red 104                                            Acetonitrile (1/1)
           5: Yello w 5       11: Red 3                                             A/B) 90/10 to 0/100 in
                                                                9                    5.5 min
300        6: Red 40          12: Red 105
                                                                                          0/100 for 1.5 min
                                                                                          90/10 for 2 min
                                                                                     and end
                                                                          Flow Rate 1.2 mL/min
200
                                                                    10    Temperatu 40 C
                                                    7                     re
                          3                                               Detection Maxplot at 300 - 600 nm
                                                                         11 12
                                   5       6
100              1 2                                                      Sample    4 L
                               4                                          Volume



  0


      0              1                 2            3          4             5           6   min
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Synthesis Color Pigments UFLC-LCMS
8.0 (x1,000,000)
                         1
                               2 3           4                                 8                   10
7.0                                                        6                                                  12
                                                 5
                                                                                                         11
6.0
                                                                                               9

                                                                           7
5.0
                                                                                                                          TIC
4.0                                                                                                                       m/z 467
                                                                                                                          m/z 537
                                                                                                                          m/z 421
3.0
                                                                                                                          m/z 407
                                                                                                                          m/z 451
2.0                                                                                                                       m/z 763
                                                                                                                          m/z 747
1.0
                                                                                                                          m/z 557
                                                                                                                          m/z 784.5
                                                                                                                          m/z 834.5
0.0                                                                                                                       m/z 972.5
      0.0          0.5       1.0       1.5           2.0       2.5   3.0           3.5   4.0       4.5        5.0   min


Detector:                            LCMS-2010EV
Column:                              Shim-pack XR-ODS (50 mmL x 2.0 mmI.D. Particle size: 2.2 µm)
Mobile phase:                        A; 20mM ammonium acetate buffer (pH 4.7)
                                     B; 20mM ammonium acetate buffer (pH 4.7)/acetonitrile = 1/1
Flow rate:                           0.5 mL/min
Injection volume:                    5 µL
Time program:                        10%B (0 min) - 100%B (5.0 min)

DUIS, negative, Scan m/z 200-1200, 1: tartrazine, 2: amaranth, 3: indigo carmine, 4: new coccine, 5: sunset yellow FCF,
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6: allura red AC, 7: fast green FCF, 8: brilliant blue FCF, 9: acid red, 10: phloxine B, 11: erythrosine, 12: rose bengal
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