Kromatografi Cair

Kinerja Tinggi
(KCKT/HPLC)
Henry K.S

HPLC

Tipe Kromatografi Cair

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

HPLC

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

Lingkup HPLC

HPLC

Efisiensi Kolom
Efek partikel size fase diam

HPLC

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
r2
Hex = 24D
M

Hex = efek ekstra kolom

r = jari-jari tubing

= laju alir linear (cm/s)

DM = koefisien difusi solute dlm fase gerak
HPLC

Efek dari sample size (g sample/g packing)

HPLC

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

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

HPLC

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 contact (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 contact (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 g/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
User Friendly
Control of HPLC Instruments

HPLC

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