Hplc n432u

Kromatografi Cair Kinerja Tinggi (KCKT/HPLC) Henry K.S

HPLC

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Tipe Kromatografi Cair Kromatografi Partisi Kromatografi Adsorpsi (LSC) Kromatografi Pertukaran Ion (IEC) Kromatografi Eksklusi Ukuran / Kromatografi Gel

HPLC

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Sejarah HPLC Sample

• Uses Glass Column filled with Calcium Carbonate PE • Diameter 1 – 5 cm • Length 50 – 500 mm A • Particle size 150 – 200 m • Flow rate 1/10 – 1 ml/min B • Separation time: hours

HPLC HPLC

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Lingkup HPLC

HPLC

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Efisiensi Kolom Efek partikel size fase diam

HPLC

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Pelebaran pita ekstra kolom - Terjadi saat solute melewati sistem injeksi, detektor dan pipa/tubing konektor - Pelebaran terjadi karena perbedaan laju alir antara fase gerak yang berada di pusat dan di tepi r2µ Hex = 24D M

Hex = efek ekstra kolom r = jari-jari tubing

µ = laju alir linear (cm/s) DM = koefisien difusi solute dlm fase gerak HPLC

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Efek dari sample size (µg sample/g packing)

HPLC

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Instrument HPLC Basic LC Hardware 1. Degasser 2. LC Pumps 3. LC Injector 4. LC Column 5. LC Detectors 6. LC Data Processor

Degasser

Extended LC Hardware 1. Column Oven

HPLC

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Modern LC Pump • Isocratic Pumps Isocratic HPLC Systems • Gradient Pumps Gradient HPLC Systems Low Pressure Gradient (LPG) Quarternay LPG High Pressure Gradient (HPG)

HPLC

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Quarternary LPG Systems • 4 Mobile Phase Bottle • On-line Vacuum Degasser 4 channel • Proportioning Valve (LPG Valve Kit) • Pumps • Dynamix Mixer

HPLC

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HPG Systems • 2, 3 or 4 Mobile Phase Bottle • Optional On-line Vacuum Degasser 2, 3, 4 channel • 2, 3 or 4 Pumps • Dynamix Mixer

HPLC

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Degassing Technique 1. Ultrasonic Degassing Non Continuous Process Low Degassing Efficiency Applicable to Isocratic Applications

2. Helium Sparging Semi Continuous Process Higher Degassing Efficiency High Operating Cost Related to Helium Gases Space Requirements and Handling

3. On-line Vaccum Degasser Continuous Process Best Degassing Efficiency Low Operating Cost Small Space Requirements Easy Handling HPLC

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Modern LC Pump • Uses Reciprocating Piston • Dual Piston Design • Pump Head SS stable up to 6000 psi (400 bar) • Optional Pump Head Titanium , Inert and stable to High Pressure • Flow Rate Ranges : 0.1 – 10.00 ml/min • High Flow Accuracy, Generally 2 % or lower • High Flow Precisions , Generally 0.2 % or lower • Low Pressure Ripple, Generally 2.5 % or lower • Microprocessor Control / PC Computer Control • Built-in Safety Features • Maintenance Data / GLP Data

HPLC

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Injector Manual Sample Injected Manually Using Syringe Fix Injected Volume, depends on Sample loops Built-in reed (trigger)

HPLC

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Auto Injector • Samples were put into vials • Vials were loaded into Racks • Auto Injector was controlled By PC Software • Variable / Programmable Injected • Volume (1 L – 500 L) • Built-in reed (trigger) Additional Features • Automatic Multiple Injections / Sample • Automatic Dilutions • Automatic Derivatizations / Reactions HPLC

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Auto sampler • • • • • • •

High End Autosampler for Analysis 96 Samples Capacity + 3 Reagents Bottle Programmable Injections Volume Multiplicate Injections / Sample Missing Vials Detections Easy Operations Programmable Mixing, Washing, Dilutions, Derivatizations • Extreemly Low Carry Over < 0.01 % • Reproducibility : < 0.3 %

HPLC

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Column Oven

Column thermostat (5-80oC)

HPLC

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HPLC Column Generally Made of SS Dimensions (75 – 300) mm L x (2 – 8) mm Dia Particle Size : 1.5 m – 10 m Stationary Phase C18 (Octadecyl) / ODS RP C8 (Octyl), C4 (Butyl) CN

NP

Anion / Cation Exchange Special GPC Column HPLC

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Detector LC Detectors Ideal Requirements • Adequate Sensitivity • Good Stability , Repeatability and Reproducibility • Linear Response Over The Wide Ranges • Short Response Time • Minimum Internal Volume For Minimum Band Broadening • Ease of Use

HPLC

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Available LC Detectors • UV-Vis Detector / Photo Diode Array Detector (PDA /DAD) • Fluoresence Detectors • Refractive Index (RI) Detector • Conductivity Detector • Mass Spectrometer Detector (LC-MS) • Electrochemical / Pulsed Amperometric • Evaporative Light Scattering Detector • Light Scattering Detector • Chiral Detector

HPLC

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LC Detectors General Summary Detector Type

Application

Detection Limit

• UV/VIS Detector:

UV absorbing substances

approx. 0.3 ng/ml

• RI Detector:

universal

approx. 0.7 µg/ml

(Refractive Index Detector)

• ELS - Detectors:

universal (Evaporative Light-Scattering Detector)

approx. 1 ng/ml

• Fluorescent detector:

fluorescent substances

approx. 0.8 pg/ml

• Conductivity detector:

ion selective

up to 0.2 % difference

• Electrochemical detector:

reducible/oxidable substances

approx. 1 pg/ml

HPLC

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Evaporative Light Scattering Detection

HPLC

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Evaporative Light Scattering Detection

HPLC

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Data Processor • Recorder

To Record Detector Signal No Possibilities to Save Data No Possibilities for Automatic Calculations Operator Dependents Results No Format Report All data has to be printed (Recording Paper)

• Integrator / Data Processor To Record Detector Signal Saved Data Limited by RAM Possible for Automatic Calculations after Run All data has to be printed (Thermal Paper) Very Limited Format Report HPLC

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Data Processor • Software

To Record / Display Detector Signal No Limitations on Saved Data Depends on HD Fully Automatic Calculations including during Acquisitions / Run Very Objective Results Very Flexible Format Report Selected Data Can Be Reported Friendly Control of HPLC Instruments

HPLC

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