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HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY
Kromatografi Cair Kinerja Tinggi (HPLC)
+Extraction
+Distillation
+Precipitation
+Chromatography
Several different separation
technique:
HPLC adalah alat yang sangat bermanfaat
dalam analisis. Bagian ini menjelaskan
bagaimana pelaksanaan dan penggunaan
serta prinsip HPLC yang sama dengan
kromatografi lapis tipis dan kromatografi
kolom
Chromatography is an analytical
chemistry techniques for the
separation of mixtures. It involves
passing the sample, a mixture which
contains the analyte, in the "mobile
phase", often in a stream of solvent,
through the "stationary phase.
Separation based on differences of
migration of each component due to the
difference of the nature (interaction)
of each component to the stationary
and, mobile phase
A,B,C
A B C
C
B
A
Mobile Phase
Stationary
phase
The stationary phase in HPLC
refers to the solid support
contained within the column
the mobile phase continuously flows
CHROMATOGRAPHY: most widely
used
+Qualitative and quantitative
+Many variety of compounds
+Good precision and accuracy
+Many choices of developing
separation
method
HPLC merupakan perkembangan tingkat tinggi dari
kromatografi kolom. Selain dari pelarut yang menetes
melalui kolom di bawah grafitasi, didukung melalui
tekanan tinggi sampai dengan 400 atm. Ini membuatnya
lebih cepat.

HPLC memungkinkan penggunaan partikel berukuran
sangat kecil untuk material terpadatkan dalam kolom yang
akan memberi luas permukaan yang lebih besar
berinteraksi antara fase diam dan molekul-molekul yang
melintasinya. Hal ini memungkinkan pemisahan yang
lebih baik dari komponen-komponen dalam campuran.

Perkembangan yang lebih luas melalui kromatografi
kolom mempertimbangkan metode pendeteksian yang
dapat digunakan. Metode-metode ini sangat otomatis dan
sangat peka.
CLASSIFICATION
Mobile Phase Gas Liquid
Samples Volatile - Less volatile
Thermally stable - Less thermally
- High molecular
weight compound
- ionic species
Collection(rec.) Less convenient Easy
Column Limited Greater variety
Interaction One phase Two phases
Operating temp. High room
temperature
Detector Limited Choice More Choices

Parameters GC LC
The differences between modern and
classical LC
Classical LC

Column used only once
Solvent flow gravity/cappilarity
Detection manually, coloring
Quantitation Less precise,
accuracy speed of
Sep Slow
Resolution low
HPLC

reusable
Pump
Continuously
More Faster


Better
MODERN VS CLASSICAL LC
Bed
Preparation
Sample
application
Solvent flow
Detection,
quantitation
Result
Color Form
TLC
HPLC
MECHANISM OF SEPARATION IN HPLC
EXCLUTION CHROMATOGRAPHY
Separation are based on a sieving process
If Solute is larger than the largest pores, it
will be excluded and smaller molecule can
enter a few pore have longer retention time.

Small M
Large BM
THF
(Molecular Sieving/GPC:
Gel Permeation
Chromatography)
Adsorption
Partition
Ion Exchange
Exclution
High performance liquid chromatography (HPLC) Methods
High performance liquid chromatography (HPLC) Methods
Fase normal HPLC

sama dengan kromatografi lapis tipis atau
kromatografi kolom. Kolom diisi partikel silika yg
sangat kecil 7 pelarut non polar misalnya heksan.
Kolom Cin 4.6 mm panjang 150 sd. 250 mm.

Senyawa-senyawa polar dalam campuran melalui
kolom akan melekat lebih lama pada silika yang
polar dibanding dengan senyawa-senyawa non polar.

Oleh karena itu, senyawa yang non polar kemudian
akan lebih cepat melewati kolom.
Fase balik HPLC( bentuk yang biasa digunakan dalam HPLC)
ukuran kolom sama, tp silika (fase diam) dimodifikasi menjadi non
polar melalui pelekatan rantai-rantai hidrokarbon panjang pada
permukaannya secara sederhana baik berupa atom karbon 8 atau 18.
Sebagai contoh, pelarut polar digunakan berupa campuran air dan
alkohol seperti metanol.

Dalam kasus ini, akan terdapat atraksi kuat antara pelarut polar dan
molekul polar dalam campuran yang melalui kolom. Atraksi yang
terjadi tidak sekuat atraksi fase diam dan molekul-molekul polar
dalam larutan. Oleh karena itu, molekul-molekul polar dalam
campuran akan menghabiskan waktunya untuk bergerak bersama
dengan pelarut.



Fase balik HPLC( sambungan)

Senyawa-senyawa non polar dalam campuran akan cenderung
membentuk atraksi dengan gugus hidrokarbon karena adanya
dispersi gaya van der Waals. Senyawa-senyawa ini juga akan
kurang larut dalam pelarut karena membutuhkan pemutusan ikatan
hydrogen sebagaimana halnya senyawa-senyawa tersebut berada
dalam molekul-molekul air atau metanol misalnya. Oleh karenanya,
senyawa-senyawa ini akan menghabiskan waktu dalam larutan dan
akan bergerak lambat dalam kolom.

Ini berarti bahwa molekul-molekul polar akan bergerak lebih cepat
melalui kolom.


CHARACTERISTIC OF NORMAL AND REVERSE PHASE


STATIONARY PHASE

MOBILE PHAS E

TYPICAL STATIONARY




TYPICAL MOBILE PHASE



NORMAL PHASE

polar

nonpolar

silica gel
alumina
hydroxyapatite


Hexane
Chloroform
iso-octane
REVERSE PHASE

nonpolar

polar

-C-18
-C-8
-Phenyl
-CN

methanol
water
acetonitryl
O
Si-OH
Si-OH
OH
Hexane
OH
C
CH
3

CH
3

CH
3

C
CH
3

CH
3

H
3
C
CH
3

NORMAL PHASE
LIQUID SOLID



Sample with
high polarity
will be
retained
Si-O-Si-C18
OH
CH3CN/H2O
OH
C
CH
3

CH
3

CH
3

C
CH
3

CH
3

H
3
C
CH
3

REVERSE PHASE
Sample with high
polarity will be eluted
easily



Reverse phase
+most widely used
+Polar, nonpolar, neutral, ionic compound

Ionic compound by reverse
phase
+ Ion suppression
+ Ion pairing
+ Ion excgange



Ion pairing

X
+
+ Y
-
XY

Ionic Sample Counter Ion neutral

HA H
+
+ A
-



Neutal PH << Ionic
ION SUPPRESSION
CH
3
CN /H
2
0
COO
-
COOH
Si-O-Si-C18
COOH
Lower pH
Benzoate ion
Benzoic acid
pH 2.5
Column : Novapak C
18

Mobile Phase : CH
3
CN/H
2
O
With PIC A pH 7.7
ION PAIRING
C
4
C
4


+
N OH
C
4
C
4



CH
3

l
N
N
l
CH
3


COO
-

ION PAIR
Benzoic Acid
N
H
3
C-N
O
O
Si-O-Si-C
18

C
4
C
4


+
N OH
C
4
C
4


Caffein
ION PAIR REAGENTS
PIC B FOR BASES
N
+

H
2
PO
4
-

SO
3
-

SO
3
-

SO
3
-

SO
3
-

STRONG BASE STRONG ACIDS
PIC A FOR ACID
Pentan Sulfonat Acid
Hexane Sulfonic Acid
Heptane Sulfonic acid
Octane Sulfonic Acid
Tetra Butyl Amonium
Phosphate
CHEMICAL STRUCTURE
ION EXCHANGE THEORY
SO
3
-
, COO
-


Cation Exchange
-(R)
2
NH
+

Anion Exchange
X
-
+ R
+
Y
-
Y
-
+ R
+
X
-
(anion exchange)
X
+
+ R
-
Y
+
Y
+
+ R
-
X
+
(Cation exchange)

Retention Time
Waktu yang dibutuhkan oleh senyawa untuk bergerak melalui kolom
menuju detektor disebut sebagai waktu retensi. Waktu retensi diukur
berdasarkan waktu pd saat sampel diinjeksikan sampai sampel
menunjukkan ketinggian puncak yang maksimum dari senyawa itu.
Senyawa-senyawa yang berbeda memiliki waktu retensi yang berbeda.
Untuk beberapa senyawa, waktu retensi akan sangat bervariasi dan
bergantung pada:
tekanan yang digunakan (karena itu akan berpengaruh pada laju alir dari
pelarut)
kondisi dari fase diam (tidak hanya terbuat dari material apa, tetapi juga
pada ukuran partikel)
komposisi yang tepat dari pelarut
temperatur pada kolom

Retention Time
the retention time (t
R
),
the time taken for the
mobile phase to pass
through the column is
called t
M
.
capacity factor = k'
A
= t
R
- t
M
/ t
M

A term called the retention factor, k', is
often used to describe the migration rate of
an analyte on a column
Column Efficiency
Column efficiency, also known as plate count, is a measure of the
dispersion of a peak. Narrow peaks take up less space in the
chromatogram and thus allow more peaks to be separated. They are also
easier to integrate since they give better resolution and less overlapping.
Efficiency is usually explained using the concept of theoretical plates.
This model supposes that the column contains a large number of separate
layers. Separate equilibrations of the sample between the stationary and
mobile phase occur in these plates.
where t is the retention time of the peak of interest and W is the
peak width at the base
Theoretical plate
Similar to the measurement
for resolution, the
measurement for efficiency
may also be performed
using the peak width at half
height, where Wh/2 is the
peak width as half height
Resolution
Resolution
Chromatogram = A plot of the detectors signal as
function of elution time or volume.
Retention time = The time a solute takes to move
from the point of injection to the detector (t
r
).
Retention volume =The volume of mobile phase
needed to move a solute from its point of injection to
the detector (V
r
).
baseline width = The width of a solutes
chromatographic band measured at the baseline (w).

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In relation with the physical parameters
expressed as follow:
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.
|

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|
k + 1
k
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.
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o
1 o
=
'
'
N
4
1
R
Selectivity efficiency
Capacity
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OPTI MI SI NG RESOLUTI ON
By MODIFYING , and N
A.
1
(RETENTION FACTOR)

CHANGE SOLVENT RATIO / MIXTURES
(SOLVENT STRENGTHS)
E.G. 50/50 MeOH/H
2
0 - 70/30 MeOH/H
2
0
B. (SELECTIVITY FACTOR)

CHANGE MOBILE PHASE CHEMISTRY
(CHANGE SOLVENT TYPES)
E.G. MeOH/H20 - THF/H20ADD
BUFFER/MODIFIER
CHANGE COLUMN TYPE
C. N (EFFICIENCY FACTOR):

CHANGE FLOW RATE (DECREASING F.R. INCREASES
EFFICIENCY)

CHANGE PARTICLE SIZE (DECREASE PARTICLE
SIZE INCREASES EFFICIENCY

CHANGE TO A LONGER COLUMN (INCREASES
COLUMN LENGHTH INCREASE EFFICIENCY)
0
0 1
V
V - V
' , tention Re = k
0 1
0 2
V - V
V - V

'
'
' , y Selectivit =
k
k
= o
1
2
2
1
1
W
V
16 N , Efficiency
|
.
|

\
|
=
The physical meaning

1
= 0 (No separation, compound elutes at void vol. (vow))

1
= 2 - 10 (recommended value)
= 1.0 (no separation)
= 2.0 (good peak separation)
N = 3000 10.000 plates / column
R = 1.5 (baseline separation)




HPLC COMPONENT
EQUIPMENT AND OPERATION
Pumps
material :stainless steel, titanium or some ceramic
materials that are resistant to corrosive
Flow rate : 0.1 ml/min to 5-10 ml/min in analytical column
(i.d. ~ 4-5 mm) deviation : less than 0.5-1%.
Reservoir : inert, mobile phase degassed , vacuum,
purge by helium or ultra sonic
Sample Injector
Chromatographic column
id are 2 to 3 mm lengths are 5-30 cm
packing material with diameter 3-10 m

Detector
Selective detector with signal proportional
to the concentration of only specific substance
in eluate
Universal (non specific) detector, with
response proportional to the a certain overall
property of the eluate, i.e. of both the solute
and the component of mobile phase. (RI)

UV/VIS detector
I
o

Log = cbc = A
I

I
o
= incident light
I = the itensity of the
transmitted light
c = Molar absortivity
b = Cell pathlength
c = Sample concentration
A = Absorbance

Fixed and Variable
Wavelength and diode
array
Data Processing Devices
Borwin sofware
Qualitative and quantitative Analysis
t
R
is characteristic of the substance with direct
comparation to standard
quantitative analysis the base on measurement of peak
heigh or peak areas value
1. TURN ON THE POWER
2. PREPARE FI LTERED AND DEGASSED SOLVENT, AND
FI LTERED SAMPLE
3.PRIME PUMP TO REMOVE BUBLE
4. ADJ UST FLOW RATE COMPI SI TI ON OF SOLVENT
AND WAVELENGH USI NG BORWI N SOFTWARE
5. EQUI LBRATE THE SYSTEM BY FOLLOWI NG THE
WORKI NG SOLVENT
6. I NJ ECT STANDARD/ SAMPLE
7. I NTEGRATE AND PRI NT CHROTOGRAM RESULT
(y-RAM SOFTWARE)


OPERATON OF HPLC
SAMPLE PREPARATION
The purpose : to clean up the sample
+ Reduce overall sample load onto column
+ Remove interference associates with matrix
+ Concentrate the component of interest
+ Enhance sensitivity
+ Remove particulate
+ Protect the column
+ I mprove the chromatographic separation peak
Type of sample clean up
+Extraction
+Filtration
+TLC
+Selective precipitation

+Open column technique
+Centrifugation
+Guard column

A. Solvent
+Have high purity, HPLC grade
+Miscible each other if using more different solvent
+pH 3,5 7,0 ( bonded phase)
+UV adsorption
+Toxicity
+Volatility

A.1. H
2
O as a solvent
+Use high quality of water (bidest)
+Prepared freshly daily
+Use appropriate containers


PRACTICAL HINT FOR AINTENANCE
A.2 Degassing
Purpose :
+To removed gasses especially O
2
and N
2

+To prevent bubble formation in pump and fluid system
+To reach great precision of retention time and quantitation

Type: Helium Spurge, Vacuum, Sonication
A.3. pH Consideration
+Reverse and normal phase 3,5 - 8.0 (8< pH
silica solubility ; PH < 2 bonded destroyed)
+Ion exchange pH: 2 - 12

A.4 Solvent miscibility
A.5 Use of buffer : To improve Resolution
High quality, fresh, avoid precipitation

C. Column
C1. Column handling
To maximize the column lifetime
+Avoid mechanical, thermal and pressure shocks
+Always increase solvent flow rates slowly (0.1- 0.5 incr.)
+Follow manufacture instruction
C2. Guard column
+Protect column from particulate and chemical contaminant
+Sample clean up
+Trace enrichment using proper guard column
B. Filtration: remove particulate
To maintain life time column and pumping system
Type : Teflon, Nylon, cellulose
C3. Column cleaning
.Reverse phase column
+Flush with 1005 Org. Solvent as MeOH for 15 min
+Contaminated non proteinaceous material
Sequence cleaning ;

Wash solvent Volume
Water 30 ml
Methanol 30 ml
THF 30 ml
Methylene chloride 30 ml
THF 30 ml
Methanol 30 ml
Water 30 ml
+Proteinaceus material : injecting 200 ul sludge of DMSO
Normal phase column

+Sequence cleaning ;

Wash solvent Volume
Isooctane 50 ml
Methylene chloride 50 ml
Methanol 50 ml
Methylene chloride 50 ml
Isooctane 50ml

+Solvent should be dry ( less than 500 ppm water)
Store the column in appropriate solvent ( Manual)
Store with end fitting connected
Protect from mechanical, thermal shock and
vibration
Flush column thoroughly prior to storage
Leaving column less 72 hours does not require
storage procedure
C4. Column storage recommendation
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Istilah-istilah
Chromatogram = A plot of the detectors signal as
function of elution time or volume.
Retention time = The time a solute takes to move
from the point of injection to the detector (t
r
).
Retention volume =The volume of mobile phase
needed to move a solute from its point of injection
to the detector (V
r
).
baseline width = The width of a solutes
chromatographic band measured at the baseline
(w).

Soal-soal