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Janto Jatiman
Analytical Instrument

Others
Spectrometry Chromatography
TOC, EDX, DTA, etc

Qualitative analysis Quantitative analysis


Quantitative analysis
Spectrometry
• Photometer
• UV-Vis-NIR Spectrophotometer (UV-Vis)
• Diode Array UV-Vis Spectrophotometer (PDA)
• RF- Spectrophotometer (RF)
• Infra Red Spectrophotometer atau (IR)
• Fourier Transform Infra Red Spectrophotometer
(FTIR)
• Atomic Absorption Spectrophotometer (AAS)
• Ion Couple Plasma Spectrophotometer (ICP)
Quantitative Analysis Spectrometer
Enzyme Reaction Analysis
Chromatography
• Gas Chromatography (GC)
• High Performance Liquids Chromatography (HPLC)
• Size Exclusion or Gel-Permeation (SEC or GPC)
• Ion Chromatography (IC)
• Thin Layer Chromatography (TLC)
• Paper Chromatography
E A

D Chromatograph

B
C
Sample: mixture of
volatile liquids (~1L)

Chromatogram

B
E
C
A
Abundance

0 5 10 15 20
Time (minutes)
Chromatogram Animation

Column
“Typical” Chromatograph
Chromatographic Principle
Solutes within a solution or volatiles within
a gas are placed in a mobile phase and
passed over a selected “adsorbent” material
(the stationary phase).
The solutes or volatiles have differential
“affinity” for the absorbent material and
thus separate. For a given system, each
compound has a “unique” retention time.
Chromatographic Principle
During separation, like molecules generally
migrate together.
However, due to a combination of natural
forces (diffusion) and equipment design
(plumbing), like molecules may separate in
what is called “band spreading.”
Representation of a chromatographic separation
Schematic of Gas Chromatography

Injection Port

Detector
Capillary Column

Data System
or Recorder

Carrier Gas Oven


Supply
Gas Chromatography - GC
Mobile phase is a gas.
Stationary phase is liquid on solid support or a
solid only.
It uses three processes to separate compounds:
a. Partition Chromatography
b. Adsorption Chromatography
c. Volatility (boiling point) Differences
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Gas Chromatography - GC
Gas Supply: Argon, Helium, Hydrogen
Impurities are removed
Injector: Pre-heated
Furnace: Should be uniformly heated
Temperature gradients are possible
Column: Usually glass capillary tube filled
with fused silica. Alumina or inert
metals are also used.
Detector: Electron capture, flame ionization, or
Mass Spectrometer
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Gas Chromatography - GC
Sample is
introduced onto a
column.

The mixture is
eluted with a
constant stream of
(usually) inert gas.

Components of the
mixture are
detected and www.tradeall.com
analyzed.
Shimadzu Gas Chromatography
Detectors available:

• FID (Flame Ionization Detector)


• TCD (Thermal Conductivity Detector)
• ECD (Electron Capture Detector)
• FTD/NPD (Flame Thermionic Detector, Nitrogen
Phosphorous Detector)
• FPD (Flame Photometric Detector)
• PID (Photoionization Detector)
• PDID (Pulse Discharge Ionization Detector)
• ELCD (Electrolytic Conductivity Detector)
• MS (Mass Spectrometric)
NPD = Flame Thermionic Detector (FTD)
Kromatografi Cair Kinerja Tinggi
HPLC

High Pressure Liquid Chromatography


or High Performance Liquid Chromatography.
Uses high pressure pumps to improve efficiency
of separation. Mobile phase is one or more
solutions and stationary phase is solid.
Used for separation, identification,
purification, and quantification.
Functional description of the instrument

• Mobile Phase reservoir, filtering


• LC Pump
• Column
• Detector
• Manual Injector/ Automatic Injector
• Column Oven
• Data processor, Recorder, Chromatopac
or PC software
HPLC Instrumentasi
Isocratic System HPLC

SPD, SPDM
RF, RID, CDD
Low Pressure Gradient HPLC
High Pressure Gradient HPLC

High Pressure Gradient HPLC


HPLC Analytical Columns

1. Packed with various


particles.
2. Are often
unidirectional.
3. Are often preceded
by a preparative or
“guard” column.
4. Usually have 3-5
mm diameter but of
micro-columns or
capillary columns
ranges from 3 µm to
200 µm.
Typical LC columns are 10, 15
and 25 cm in length and are
fitted with extremely small
diameter (3, 5 or 10 mm)
particles. The internal
diameter of the columns is
usually 4 or 4.6 mm; this is
considered the best
compromise among sample
capacity, mobile phase
consumption, speed and
resolution. However, if pure
substances are to be
collected (preparative scale),
larger diameter columns may
be needed
JUMLAH PELAT TEORITIS, N
Rt Formula :
N = 16 x ( Rt / W )2

Area Modifikasi formula pada


pengukuran sebenarnya :
H N = 5.54 x ( Rt / W 1/2 )2
W1/2
H1/2 Modifikasi formula pada
integrator :
W N = 6.28 x (Rt x H / Area)2
JUMLAH PELAT TEORITIS, N
 Ketiga formula benar hanya jika
puncak Gaussian-shaped.
 untuk puncak asimetri :
(tR/W0.1)2
N = 41.7 T + 1.25

dimana W0.1 = peak width pada 10% tinggi


dan T = tailing factor
JUMLAH PELAT TEORITIS

 N menunjukkan efisiensi kolom.

N=15000 N=3000

Efisiensi kolom baik Efisiensi kolom buruk


Detector HPLC
• SPD UV-Vis Spectrophotometer
» Fixed wavelength
» Variable wavelength

• SPDM Photodiode array UV-Vis Detector


• RID Refractive Index Detector
• RF Fluorescence Detector
• CDD Conductivity Detector
• ELSD Evaporative Light Scattering Detector
• MS Mass Spectrometric
“Typical” Chromatograph
Co-Sense for BA

Automate sample pretreatment for higher


productivity.
Innovative, efficient analysis of bio-samples.
1. A simple and rapid method.

2. Used to monitor purity of organic


compounds.

3. Mobile phase is a solvent and the


stationary phase is a solid adsorbent
on a flat support (often silica gel).
TLC
Size Exclusion or Gel-Permeation
protein
salt ions
molecule

SEC or GPC buffer

Mobile phase is a solvent.


gel
particle

Stationary phase is a packing


of porous particles.
Apakah SEC ?

• Size Exclusion Chromatography


(SEC)
– GPC (Gel Permeation Chromatography)
• terutama untuk lingkungan polimer
– GFC (Gel Filtration Chromatography)
• terutama untuk lingkungan biologi
Prinsip SEC
• Tidak ada kekuatan interaksi
• Perbedaan waktu tempuh
What Factors Are Used in Selecting
a Procedure?
Cost, speed, ease, safety, accuracy,
precision, sensitivity, and freedom from
interference.

However, to achieve this, we must select


an appropriate mobile phase, stationary
phase (column), detector, and instrument
variables (column temperature, flow
rates, etc.).
• Terima kasih

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