HPLC 1

Kromatografi Cair Kinerja Tinggi (KCKT/HPLC)

Henry K.S

HPLC 2

Tipe Kromatografi Cair

Kromatografi Partisi

Kromatografi Adsorpsi (LSC)

Kromatografi Pertukaran Ion (IEC)

Kromatografi Eksklusi Ukuran / Kromatografi Gel

HPLC 3

Sejarah HPLC• Uses Glass Column filled with

Calcium Carbonate• Diameter 1 – 5 cm• Length 50 – 500 mm• Particle size 150 – 200 m• Flow rate 1/10 – 1 ml/min• Separation time: hours

HPLC

SamplePE

A

B

HPLC 4

Lingkup HPLC

HPLC 5

Efisiensi Kolom

Efek partikel size fase diam

HPLC 6

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

Hex =r2µ24DM

Hex = efek ekstra kolom

r = jari-jari tubing

µ = laju alir linear (cm/s)

DM = koefisien difusi solute dlm fase gerak

HPLC 7

Efek dari sample size (µg sample/g packing)

HPLC 8

Instrument HPLC

1. Degasser2. LC Pumps3. LC Injector4. LC Column5. LC Detectors6. LC Data Processor

Basic LC Hardware

Extended LC Hardware

1. Column Oven

Degasser

HPLC 9

• Isocratic Pumps

Modern LC Pump

Isocratic HPLC Systems

• Gradient Pumps

Gradient HPLC Systems

Low Pressure Gradient (LPG)Quarternay LPG

High Pressure Gradient (HPG)

HPLC 10

Quarternary LPG Systems

• 4 Mobile Phase Bottle• On-line Vacuum Degasser

4 channel • Proportioning Valve (LPG

Valve Kit)• Pumps• Dynamix Mixer

HPLC 11

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 12

Degassing Technique

1. Ultrasonic Degassing

2. Helium Sparging

3. On-line Vaccum Degasser

Non Continuous ProcessLow Degassing Efficiency Applicable to Isocratic Applications

Semi Continuous ProcessHigher Degassing Efficiency High Operating Cost Related to Helium GasesSpace Requirements and Handling

Continuous ProcessBest Degassing Efficiency Low Operating CostSmall Space Requirements Easy Handling

HPLC 13

• Uses Reciprocating Piston

Modern LC Pump

• 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 14

Injector Manual

Sample Injected Manually Using SyringeFix Injected Volume, depends on Sample loops Built-in reed contact (trigger)

HPLC 15

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 contact (trigger)

Additional Features

• Automatic Multiple Injections / Sample• Automatic Dilutions• Automatic Derivatizations / Reactions

HPLC 16

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 17

Column Oven

Column thermostat (5-80oC)

HPLC 18

HPLC Column

Generally Made of SS Dimensions (75 – 300) mm L x (2 – 8) mm DiaParticle Size : 1.5 m – 10 m

Stationary Phase

C18 (Octadecyl) / ODSC8 (Octyl), C4 (Butyl)CN

RP NP

Anion / Cation Exchange GPC ColumnSpecial

HPLC 19

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 20

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 21

LC Detectors General Summary

Detector TypeDetector Type ApplicationApplication Detection LimitDetection Limit

• • UV/VIS Detector:UV/VIS Detector: UV absorbing substances approx. 0.3 ng/ml

• • RI Detector:RI Detector: universal approx. 0.7 µg/ml (Refractive Index Detector)

• • ELS - Detectors:ELS - Detectors: universal approx. 1 ng/ml(Evaporative Light-Scattering Detector)

• • Fluorescent detector:Fluorescent detector: fluorescent substances approx. 0.8 pg/ml

• • Conductivity detector:Conductivity detector: ion selective up to 0.2 % difference

• • Electrochemical Electrochemical reducible/oxidable approx. 1 pg/ml

detector:detector: substances

HPLC 22

Evaporative Light Scattering Detection

HPLC 23

Evaporative Light Scattering Detection

HPLC 24

Data Processor

• Recorder

• Integrator / Data Processor

To Record Detector SignalNo Possibilities to Save DataNo Possibilities for Automatic CalculationsOperator Dependents ResultsNo Format ReportAll data has to be printed (Recording Paper)

To Record Detector SignalSaved Data Limited by RAMPossible for Automatic Calculations after RunAll data has to be printed (Thermal Paper)Very Limited Format Report

HPLC 25

Data Processor

• Software To Record / Display Detector SignalNo Limitations on Saved Data Depends on HDFully Automatic Calculations including during Acquisitions / RunVery Objective ResultsVery Flexible Format ReportSelected Data Can Be ReportedUser Friendly Control of HPLC Instruments