Analisis Elektroforesis Gel Dan CE TM 13

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PRINSIP ELEKTROFORESIS
Faktor yang menyebabkan pemisahan MK. ANALISIS ELEKTROKIMIA
 Muatan molekul Fachrurrazie, M.Si
ELEKTROFORESIS
 Bobot molekul
 Bentuk molekul
Molekul dipengaruhi oleh medan listrik
V = (EQ)/f
MK. ANALISIS ELEKTROKIMIA
V = molecule velocity
Fachrurrazie, M.Si
E = Eletric field strength
Q = molecular charge
F = friction coefficient of molecule
 Hasil pemisahan yang tinggi sangat tergantung berbagai faktor
1
seperti rumus tersebut 2

Fachrurrazie, M.Si
THEORY OF ELECTROPHORESIS APLIKASI ELEKTROFORESIS GEL

The movement of a charged molecule subjected to an
electric field is represented by the following equation: MK. ANALISIS ELEKTROKIMIA
Fachrurrazie, M.Si
 Advantages
Eq Analysis of carbohydrates  Fast
V =  Small Sample
f Analysis of inorganic
 Relatively inexpensive
V: the velocity of the molecule anions/metal ions  Automated
E: the electric field in volts/cm  Disadvantages
DNA profiling  Cannot identify neutral species
q: the net charge on the molecule (Kecuali yang osmosis)
f: frictional coefficient, which depend on the Protein identification  Joule Heating

mass and shape of the molecule  Cannot discern shape
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MK. ANALISIS ELEKTROKIMIA MK. ANALISIS ELEKTROKIMIA

Fachrurrazie, M.Si Fachrurrazie, M.Si
MIGRASI SAMPEL ELEKTROFORESIS DAN BUFFER

45kD 10kD 25kD
 Beberapa jenis larutan yang dianjurkan untuk electro phoresis DNA
Katode adalah;TAE (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA).
 Fragmen DNA akan migrasi dengan kecepatan yang berbeda dalam
kedua dapar tersebut dan sangat tergantung kekuatan ion.
 Buffer tidak saja untuk mempertahankan pH, tetapi memberikan ion
yang membantu sifat konduktivitas
 Bila digunakan kadar dapar 10 x lebih kuat dari larutan stok, suhu
dapat naik pada proses elektroforesis gel dapat meleleh
Anode
5 6
IN MOST ELECTROPHORESIS UNITS, THE GEL IS MOUNTED BETWEEN TWO BUFFER

CHAMBERS CONTAINING SEPARATE ELECTRODES SO THAT THE ONLY ELECTRICAL
CONNECTION BETWEEN THE TWO CHAMBERS IS THROUGH THE GEL. TUBE GEL UNITS
Fachrurrazie, M.Si
DC
Cathode Applied Anode
Potential
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SLAB GEL UNIT FLAT BED UNIT MK. ANALISIS ELEKTROKIMIA

Fachrurrazie, M.Si
Fachrurrazie, M.Si
CONTINUOUS AND DISCONTINUOUS

BUFFER SYSTEMS PREPARASI GEL ELECTROPHORESIS

Fachrurrazie, M.Si
12
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PROSES PEMISAHAN GEL INTERRELATION OF RESISTANCE,

ELECTROPHORESIS UNTUK WESTERN BLOT
VOLTAGE, CURRENT AND POWER
 Two basic electrical equations are important in
electrophoresis
 The first is Ohm's Law, I = E/R
 The second is P = EI
 This can also be expressed as P = I2R
 In electrophoresis, one electrical parameter, either current,

voltage, or power, is always held constant
13 Fachrurrazie, M.Si
THE NET CHARGE IS DETERMINED

CONSEQUENCES BY THE PH OF THE MEDIUM
 Proteins are amphoteric compounds, that is, they
 Under constant current conditions (velocity is directly
contain both acidic and basic residues
proportional to current), the velocity of the molecules is
maintained, but heat is generated.  Each protein has its own characteristic charge
 Under constant voltage conditions, the velocity slows, but no properties depending on the number and kinds of
additional heat is generated during the course of the run amino acids carrying amino or carboxyl groups
 Under constant power conditions, the velocity slows but  Nucleic acids, unlike proteins, are not amphoteric.
heating is kept constant They remain negative at any pH used for
electrophoresis
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TEMPERATURE AND ELECTROPHORESIS WHAT IS THE ROLE OF THE SOLID

SUPPORT MATRIX?
Important at every stage of electrophoresis It inhibits convection and diffusion, which
During Polymerization would otherwise impede separation of
 Exothermic Reaction molecules
 Gel irregularities It allows a permanent record of results
 Pore size through staining after run
During Electrophoresis
 Denaturation of proteins It can provide additional separation through
 Smile effect molecular sieving
 Temperature Regulation of Buffers
AGAROSE AND POLYACRYLAMIDE AGAROSE AND POLYACRYLAMIDE
Although agarose and polyacrylamide differ greatly in their physical Because the pores of an agarose gel are large, agarose is
and chemical structures, they both make porous gels.
used to separate macromolecules such as nucleic acids,
 A porous gel acts as a sieve by retarding or, in some cases, by large proteins and protein complexes
completely obstructing the movement of macromolecules while
Polyacrylamide, which makes a small pore gel, is used to
allowing smaller molecules to migrate freely.
separate most proteins and small oligonucleotides.
 By preparing a gel with a restrictive pore size, the operator can
Both are relatively electrically neutral
take advantage of molecular size differences among proteins
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AGAROSE GELS STRUCTURE OF THE REPEATING UNIT OF

AGAROSE, 3,6-ANHYDRO-L-GALACTOSE
 Agarose is a highly purified uncharged polysaccharide derived from agar Fachrurrazie, M.Si
 Agarose dissolves when added to boiling liquid. It remains in a liquid

Basic disaccharide
state until the temperature is lowered to about 40° C at which point it
repeating units of agarose,
gels
G: 1,3-β-d-galactose
 The pore size may be predetermined by adjusting the concentration of and
agarose in the gel A: 1,4-α-l-3,6-
 Agarose gels are fragile, however. They are actually hydrocolloids, and anhydrogalactose
they are held together by the formation of weak hydrogen and

hydrophobic bonds

Fachrurrazie, M.Si
GEL STRUCTURE OF AGAROSE POLYACRYLAMIDE GELS
 Polyacrylamide gels are tougher than agarose gels
 Acrylamide monomers polymerize into long chains that are

covalently linked by a crosslinker
 Polyacrylamide is chemically complex, as is the production

and use of the gel

Fachrurrazie, M.Si
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CROSSLINKING ACRYLAMIDE CHAINS CONSIDERATIONS WITH PAGE
Preparing and Pouring Gels

Determine pore size
 Adjust total percentage of acrylamide
 Vary amount of crosslinker
Remove oxygen from mixture
Initiate polymerization
 Chemical method
MK. ANALISIS ELEKTROKIMIA  Photochemical method MK. ANALISIS ELEKTROKIMIA

CONSIDERATIONS WITH PAGE SDS GEL ELECTROPHORESIS
 Analysis of Gel  In SDS separations, migration is determined not by intrinsic electrical

charge of polypeptides but by molecular weight
 Staining or autoradiography followed by densitometry
 Sodium dodecylsulfate (SDS) is an anionic detergent which denatures
 Blotting to a membrane, either by capillarity or by
secondary and non–disulfide–linked tertiary structures by wrapping
electrophoresis, for nucleic acid hybridization,
around the polypeptide backbone. In so doing, SDS confers a net negative
autoradiography or immunodetection charge to the polypeptide in proportion to its length
 When treated with SDS and a reducing agent, the polypeptides become
rods of negative charges with equal “charge densities" or charge per unit
MK. ANALISIS ELEKTROKIMIA length. MK. ANALISIS ELEKTROKIMIA
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SDS GEL ELECTROPHORESIS

Fachrurrazie, M.Si
CONTINUOUS AND DISCONTINUOUS
BUFFER SYSTEMS
 A continuous system has only a single separating gel and uses

the same buffer in the tanks and the gel
 In a discontinuous system a nonrestrictive large pore gel, called a

stacking gel, is layered on top of a separating gel
 The resolution obtainable in a discontinuous system is much

greater than that obtainable in a continuous one. However, the
continuous system is a little easier to set up
Fachrurrazie, M.Si
DETERMINING MOLECULAR WEIGHTS

OF PROTEINS BY SDS-PAGE ISOELECTRIC POINT
 Run a gel with standard proteins of known

There is a pH at which there is no net charge on a
molecular weights along with the polypeptide to be
characterized protein; this is the isoelectric point (pI).
 A linear relationship exists between the log10 of the
molecular weight of a polypeptide and its Rf
Above its isoelectric point, a protein has a net
 Rf = ratio of the distance migrated by the molecule negative charge and migrates toward the anode in an
to that migrated by a marker dye-front
electrical field.
 The Rf of the polypeptide to be characterized is
determined in the same way, and the log10 of its Below its isoelectric point, the protein is positive and
molecular weight is read directly from the standard
migrates toward the cathode. MK. ANALISIS ELEKTROKIMIA
curve Fachrurrazie, M.Si
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ISOELECTRIC FOCUSING
Fachrurrazie, M.Si
BLOTTING TECHNIQUES
Northern Western
Southern Blot
Blot Blot
 Isoelectric focusing is a method in which proteins are separated in a
DNA RNA Protein
pH gradient according to their isoelectric points Macromolecules on
the blot
 Focusing occurs in two stages; first, the pH gradient is formed Labeled DNA Labeled DNA Labeled antibodies
Probe
 In the second stage, the proteins begin their migrations toward the
Radioactively or Radioactively or Radioactively or
anode if their net charge is negative, or toward the cathode if their fluorescent labeled fluorescent labeled fluorescent
deoxynucleotide deoxynucleotide labeled amino
net charge is positive Source of labels
acids
 When a protein reaches its isoelectric point (pI) in the pH gradient,

it carries a net charge of zero and will stop migrating MK. ANALISIS ELEKTROKIMIA
Fachrurrazie, M.Si
TISSUE DISTRIBUTION OF MESSENGER A PROTEIN CAN BE SPECIFICALLY RECOGNIZED

RNA REVEALED BY NORTHERN BLOT BY AN ANTIBODY IN A WESTERN BLOT
M 1 2 3 4 M 1 2 3 4
1: heart 9 : spleen
2: brain 10: thymus
3: placenta 11: prostate
4: lung 12: testis
1 2 3 4 5 6 7 8 5: liver 13: ovary
6: skeletal 14: small intestine
muscle 15: colon
7: kidney 16: leukocyte
8: pancreas
Coomassie Blue Dye Western blot
9 10 11 12 13 14 15 16 Stained protein gel
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GEL ELECTROPHORESIS OF DNA

ELECTROFORESIS WESTERN BLOT Wells for
Direction for
DNA migration Anode
(+)
sample
loading
Cathode (- Agarose slab gel

) submerged in buffer
• Agarose is a polysaccharide derived from seaweed, which forms a

solid gel when dissolved in aqueous solution.
• When an electric field is applied to an agarose gel in the presence of
a buffer solution which will conduct electricity, DNA fragments move
through the gel towards the positive electrode (DNA is highly
negatively charged) at a rate which is dependent on its size and
shape.
Fachrurrazie, M.Si
GEL ELECTROPHORESIS OF DNA GEL ELECTROPHORESIS OF DNA

For linear DNA molecules, they have uniform • The DNA can be stained by the inclusion of ethidium
shape and charge to mass ratio. The bromide in the gel, or by soaking the gel in a solution
electrophoretic mobility of the DNA molecule of ethidium bromide after electrophoresis.
is influenced primarily by the molecular size: • The DNA shows up as an orange band on illumination
The larger molecules are retarded by the by UV light. Alternatively, methylene blue can be used
molecular sieving effect of the gel, and the to stain DNA.
small molecules have greater mobility. • Gels composed of polyacrylamide can separate DNA
molecules that differ in length by only one nucleotide
and are used to determine the base sequence of DNA.
• Agarose gels are used to separate DNA fragments that
MK. ANALISIS ELEKTROKIMIA have larger size differences. MK. ANALISIS ELEKTROKIMIA
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PROCEDURES OF DNA FINGERPRINTING

PROCEDURES OF DNA FINGERPRINTING
• In order to detect specific sequences, DNA is usually
transferred to a solid support, such as a sheet of
nitrocellulose or nylon paper.
• The paper is treated with an alkaline solution to
denature DNA, that is, separate the two strands of
each double helix.
• The single-stranded DNA can be hybridized with a
probe, and the regions on the nitrocellulose blot
containing DNA that base-pairs with the probe can be
identified.

DNA POLYMORPHISMS DETECTION OF POLYMORPHISM

Restriction Fragment Length Polymorphisms
• Polymorphisms are variations in DNA sequences.
There may be millions of different polymorphisms in • Occasionally, a point mutation occurs in a recognition
the human DNA. site for a restriction enzyme. The enzyme, therefore,
can cut at other recognition sites but not at the site of
• Polymorphisms in the human DNA serve as the basis the mutation. Consequently, the restriction fragment
for the diagnosis of diseases and the identity of produced by the enzyme is larger for a person with the
individuals. mutation than for a normal person.
• Mutations can also create restriction sites that are not
present in the normal gene. In this case, restriction
fragments will be smaller for the person with the
mutation than for the normal individual. These
variations in the length of restriction fragments are
known as restriction fragment length polymorphisms
MK. ANALISIS ELEKTROKIMIA (RFLPs). MK. ANALISIS ELEKTROKIMIA
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APPLICATION OF DNA FINGERPRINTING MUTATION HIGHLY VARIABLE REGIONS

DETECTION
• Human DNA contains many sequences that
are repeated in tandem a variable number of
times at certain loci in the genome.
• These regions are called hypervariable

regions because they contain a variable
number of tandem repeats (VNTR).

APPLICATION OF DNA FINGERPRINTING FORENSIC

DETECTIONOF HIGHLY VARIABLE REGIONS ANALYSIS
• Digestion with restriction This restriction fragment
enzymes that recognize
technique has been called
sites which flank the VNTR
region produces fragments "DNA fingerprinting" and is
containing these loci, which gaining widespread use in
differ in size from one forensic analysis. Family
individual to another, relationships can be
depending on the number of determined by this method,
repeats that are present. and it can be used to convict
suspects in criminal cases.
• Probes used to identify Individuals who are closely
these restriction fragments
related genetically will have
bind to or near the sequence
that is repeated. restriction fragment pattern
that are more similar than
those who are more distantly
related.
Fachrurrazie, M.Si
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Assessing DNA Quality Pengaruh Konsentrasi Agarose
Time
minutes seconds
Experiment:  Dalam praktek pemisahan DNA
3 2 1 45 30 15 0
• 100 ng K562 DNA dipengaruhi oleh ketebalan fase diam
• Digest with DNAse (media elektro foresis) maupun BM dari
~23Kbp sampel.
 Slide (6)mengalami penguraian ternyata
Molecular Weight Ladder
migrasi lebih cepat dibanding dengan
~ 2kbp
sebelum penguraian.
 Slide (8), menunjukkan agrose (media
Fachrurrazie, M.Si
elekt.) yang kadar tinggi memperlambat
migrasi
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AGAROSE GEL ELECTROPHORESIS

Fachrurrazie, M.Si
Acrylamide Gel Electrophoresis Fachrurrazie, M.Si
PENGARUHPERSENAGAROSE GEL DAN SIZE SEPARATION Pengaruh Persen Akrilamida Gel dan Size Separation
Agarose (%) Range of separation of linear DNA % Acrylamide (w/v) Effective range of
(Kilobases)
with BIS at 1:20 separation - bp
0.3 60 - 5
0.6 20 - 1
3.5 1 000 - 2 000
0.7 10 - 0.8
5.0 80 - 500
0.9 7 - 0.5 8.0 60 - 400
1.2 6 - 0.4 12.0 40 - 200
1.5 4 - 0.2 15.0 25 - 150
2.0 3 - 0.1 20.0 6 - 100
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Fachrurrazie, M.Si
Fachrurrazie, M.Si
II. PROTEIN STAINING PROTEIN STAINS YANG BIASA DIGUNAKAN
1. Pewarnaan Coomasie blue
Stain Detection limit Comment

Ponceau S 1-2 mg Reversible
2. Pewarnaan Dengan AgNO3 (Silver stain) Amido Black 1-2 mg Permanent; low background
Coomassie Blue 1.5 mg Permanent; high background
India Ink 100 ng Permanent
Silver stain 10 ng Permanent
Colloidal gold 3 ng Permanent

MK. ANALISIS ELEKTROKIMIA Fachrurrazie, M.Si
Fachrurrazie, M.Si
III. DNA STAINING: ETHIDIUM BROMIDE Pewarnaan sampel dengan Ethidium Bromide
 Senyawa mempunyai gugus planar yang dapat ber

intercalates diantara base DNA.
 Sifat tersebut menyebabkan kenaikan sifat flouresensinya
dibanding senyawa dalam keadaan bebas (dalam larutan).
 Ethidium bromide bergabung
dengan DNA double helix  Radiation U.V. pada 254 nm diabsorbed oleh DNA dan
and fluoresced bila disinari ditransmisikan kepada zat warna yang terikat, dan dapat
dengan UV mengemisikan warna pada 590 nm (orenye kemerahan).
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ETHIDIUM BROMIDE Electrophoresis of Nucleotides
 Ethidium bromide biasanya dibuat dengan larutan 10

mg/ml. Disimpan dan terlindung (kedap cahaya). DNA mempunyai mutan
negatif ( karena kerangka
 Zat warna kurang berinteraksi atau incorporated kedalam gel dan phosphate )
dapar yang digunakan. Tetapi gel dapat berwarna setelah
dikocok dengan ethidium bromide (0.5 ug/ml selama 30 min). dan DNA akan tertarik pada
dielusi dengan larutan. muatan positif baik dari
elektrode maupun dapar,
 Warna akan tampak bila disinari dengan UV dan dapat dipotret
dengan film polaroid. sebaliknya akan ditolak
oleh muatan negatif.
 Kepekaan DNA terhadap zat warna ini bila kadar DNA minimal 0.1
ug of DNA.

PEWARNAAN GEL CONTOH HASIL ETHIDIUM-STAINED GEL

PHOTOGRAPHED PADA SINAR UV
**Each band that you see is a collection of millions of

DNA molecules, all of the same length!!
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Fachrurrazie, M.Si
MOBILITAS FRAGMEN DNA DALAM SEL AGAROSE TIPE DARI POLYACRYLAMIDE GELS
 Ada hubungan yang linier antara Denaturing gels : Gel ini mengalami polimeri sasi
kecepatan migration setiap fragmen dengan denaturant, terutama yang bersifat dapat
DNA dan logaritma dari ukuran berpasangan dengan asam nucleat seperti urea,
molekul (in basepairs). atau formaid. MK. ANALISIS ELEKTROKIMIA
Fachrurrazie, M.Si
 Molekul yang besar bergerak lambat

karena pengaruh friksi yang lebih Denatured DNA Yang telah terdenaturasi akan
besar Bila digambarkan dengan migrasi melewati gel poliakrilamid tak terjadi
kurva sebagai berikut: reaksi (independen) atau dapat bergantian.
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METODE FLUORESCENT LABELING DNA AMINE REACTIVE DYES UNTUK LABELING DNA
FAM (Blue) JOE (Green) TAMRA (Yellow) ROX (Red)
• Intercalating Dyes (post-PCR =polimeric chain

reaction))
• Dye-labeled nucleotide insertion during PCR
• Dye-labeled primer insertion during PCR
3’TGCATCTACGATGTAATCG5’
CGTAGCTG3’
Emission 520 Emission 548 Emission 580 Emission 605
• Linkers, dyes, etc. can be added to
• the 5’ end of the primer without disturbing the
reaction.
NH2 + NH O N O
• Individual bases can also be tagged
O N O
• ENZYME Intercalation process
• Covalent labeling process Pewarna
Pewarna
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Fachrurrazie, M.Si
Dye Migration in Different % Polyacrylamide Gels Elektroforesis Kapiler
 Elektroforesis yang berlangsung pada tabung kapiler,

% Polyacrylamide gel Bromophenol blue Xylene cyanol (41)
dikenal sebagai capillary electroporesis (CE)
5 35 130
 Gabungan GC dengan elektroforesis
6 26 106
 Metode alternatif untuk kromatografi
8 19 76
Elektrokromatografi
10 12 55
 Tabung kapiler Kolom pada kromatografi gas
12 8 28
 Ukuran tabung: panjang 10-100 cm; diameter internal
25-100 m
Fachrurrazie, M.Si

Instrumentasi Elektroforesis Kapiler CAPILARY ISOELECTRIC FOCUSING
 Sebelum terjadi pemisahan analit dikumpulkan dulu , kemudian dengan

Kolom kapiler
membe rikan arus listrik terjadilah perbedaan potensial, sehingga terjadi
pengelompokan iom positif dan negatif.
 Dengan demikian pemberian potensial dapat diatur agar pemisahan dapat
mencapai optimal.
 TEKNIK INJEKSI:
 1). Hidrostatik, dengan tekanan cairan
 Besarnya tekanan tergantung volume cairan dirumuskan:
g htINJ  =Bobot Jenis
 VINJ = —————— h= Selisih Tinggi Larutan
 8L g = Gaya Gravitasi
  =Viskositas
 t= Lamanya Penyuntikan
 V= volume
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Contoh gambar Injeksi Bagan Elektroforesis Kapiler
SUSUNAN ALAT

Fachrurrazie, M.Si Aplikasi IEF
Isoelectric focusing (IEF) IEF (Isoelectric focusing)berguna untuk menentukan pI dari
protein, terutama dalam memisahkan immunoglobulin ,
Perpindahan (pergerakan) cairan pada pipa variasi hemoglobin dan menentukan posisi translational
kapiler akan terus berlangsung sepanjang larutan modifikasi dari protein rekombinan, separasi campuran
memiliki muatan atau diberi muatan, apabila protein dapat dilihat pada gambar dibawah:
kondisi sudah menjadi netral maka cairan akan
berhenti bergerak pada pH tti. (PI)

Fachrurrazie, M.Si
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APLIKASI IEF SAMPLE INJECTION IN CE
Pemisahan DNA Hydrodynamic injection Electrokinetic injection

Pemisahan oligonucleotida dan uses a pressure difference between the
uses a voltage difference between the two
sekuen produk DNA melibatkan two ends of the capillary
ends of the capillary
agar polyacrylamide, gambar Pd4 t
Vc = Qi = Vapp( kb/ka)tr2Ci
dibawah menunjukkan hasil 128Lt
separasi Q, moles of analyte
Vc, calculated volume of injection
P, pressure difference vapp, velocity
d, diameter of the column
t, injection time t, injection time
, viscosity kb/ka ratio of conductivities (separation
buffer and sample)
r , capillary radius
Ci molar concentration of analyte

Capillary Isoelectric Focusing (CIEF) Capillary Isotachophoresis (CITP)
 Capillary Isoelectric Focusing (CIEF) allows amphoteric  Capillary Isotachophoresis (CITP) adalah bedasar
molecules, such as proteins, to be separated by perbedaan sampel mana yang leading (pendahulu) dan
electrophoresis in a pH gradient generated between the mana yang termiting (yang akir) dari suatu elektrolit.
cathode and anode.
 A solute will migrate to a point where its net charge is zero.  Analit yang intermediate akan terletak diantara
At the solute’s isoelectric point (pI), migration stops and the pendahulu dan yang akir dari analit, dengan bentuk
sample is focused into a tight zone. tajam dan terfocus (focused Zone).
 In CIEF, once a solute has focused at its pI, the zone is
mobilized past the detector by either pressure or chemical  Walaupun ini digunakan untuk model pemisahan tetapi
means. This technique is commonly employed in protein sebelumnnya merupakan pemusatan dari sampel lebih
characterization as a mechanism to determine a protein's dulu.
isoelectric point.
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A. Micell Electroforesis B. Open Tubular Capilary Electrophoresis / Capilary

 Kemampuan senyawa berpartisipasi kedalam misel sangat Zone Electrophoresis (CZE)
tergantung sifat hidofobiknya
Gerakan analitnya berupa pita, terbuka karena bagian
(-) (+) tengah kapiler hanya ada cairan netral, sedangkan
didinding bagian tepi terdapat fase medium
Senyawa yang dapat dianalisis dengan cara ini antara
lain:morfin, elektroforesis, hasilnya kurang bagus karena
kanabinol, barbi turat. Benzodiazepin, antibiotika dan
vitamin penghantaran listrik kurang sempurna.
Waktu retensi dirumuskan:
k’ =PmwV[(S)-CMC)
 k’= waktu retensi, Pmw= koefisien partisi,
 S= kadar surfaktan, CMC = medium
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C. CAPILLARY ISOTACKHOPHORETIK  CONTOH s+ < BGE

Background Electrophoresis (BGE)
Dalam kapiler disuntikkan back ground elec trophoresis
(bge),dengan keceapatan bge yang jauh lebih besar dari  s+ > t
kecepatan migrasi sampel yang ionik. (S).Setelah sampel `
disun tikkan, dan diberi arus listrik maka akan terjadi migrasi `
bersama.
 Gambar tahapan peristiwa dalam kapiler sebelum ada

D. Capillary isotackhophoretik arus dan setelah diberi arus listrik.
CIF Sebelum terjadi pemisahan analit dikumpulkan dulu ,  Kecepatan migrasi sangat tergantung akan muatan, dan bobot
molekul.
kemudian dengan memberikan arus listrik terjadilah  Elektroforesis dengan gel (Capillary Gel Electrophoresis), peristiwa
yang terjadi sama dengan CITP.
perbedaan potensial, sehingga terjadi pengelompokan iom
positif dan negatif 80
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ANALISIS METADON DAN ISOMERNYA

 Metadon (MTD), sebagai analgesi yang sintetik, biasanya
digunakan untuk obat pengganti opiat yang tidak adiktif. Obat
ini mempunyai atom karbon asimitris sehingga terdapat
beberapa isomer seperti:
 R)-MTD., (S)-MTD, (S)-MTD [2,3]. Dan hasil metabolisme
menjadi 2- ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine
(EDDP), and to 2-ethyl-5-methyl-3,3-diphenylpyrrolidine
(EMDP).
 Electropherogram of an oral fluid sample spiked with 10 ng/mL (R)-PEA,used as
 Untuk pemisahan yang optimum , dengan elektroforesis internal standard (IS), obtained under optimized conditions: fused-silica capillary
kapiler (EK) digunakan fase diam highly sulphated
gammacyclodextrin (HS--CD) (Rudaz et al, 2003). 375 m o.d., m i.d., 40.2 cm total length (effective length 32.8 cm);
 Sebagai baku internal (standard internal), R)-(+)-1-  50mM phosphate buffer, pH 4.5, 0.2% HS--CD (w/v); applied voltage, 20 kV;
phenylethylamine ((R)-PEA, yang dalam EK, senyawa ini temperature: 20 ◦C, UV detection at 200 nm. MK. ANALISIS ELEKTROKIMIA
kecepatan migrasinya tidak sama dengan sampel uji; Fachrurrazie, M.Si
81 82
ANALYSIS PROTEINRAMBUT
Electropherogram of an oral fluid sample spiked with 50 ng/mL of

(R,S)-MTD, (R,S)-EDDP and (R)-PEA (IS) using the same optimized
conditions as in Electrophoresis 1998, 19, 42-50
Fachrurrazie, M.Si MK. ANALISIS ELEKTROKIMIA
Fachrurrazie, M.Si
3/4/2023

Fachrurrazie, M.Si
Separation of alkaline, alkaline earths, and Lanthanides

(from C.A.Lucy and T.L.Mcdold,Anal.Chem.1995,67,1074.)
Capillary: 36.5 cm 75 µm fused silica, 30 kV .

Separation of natural isotopes of 0.56 mM Cl- by capillary
electrophoresis with indirect spectrophotometric detection at 254 Detection: indirect UV, 214 nm.
nm.
(D.A.Skoog,F.J.Holler,T.A.Nieman,Principles Of Instrumental Analysis,Fifth
MK. ANALISIS ELEKTROKIMIA Edition,Sanders Coliege Publishing,1998,778)
Fachrurrazie, M.Si
Pemisahan kation MK. ANALISIS ELEKTROKIMIA

Fachrurrazie, M.Si
S: system peak; Model Proteins

separated
1: ammonium;
2: potassium;
3: calcium;
Model Proteins separated at pH= 2.7
4: sodium; Peak No. Proteins mol. Wt. Isolelectric Point, PI
5: magnesium; 1 Cytochrome c 12,400 10.7

2 Lysozyme 14,000 10.1
6: zinc 3 Trypsin 24,000 10.1
(Y. Fung, K. Lau, H. Tung, Talanta, 45, 1998, 619.)
4 Trypsinogen 23,700 8.7
5 Trypsin inhibitor 20,100 4.5
Fachrurrazie, M.Si
3/4/2023

(H.B.Wan,J.Liu,Talanta,45,1998,663)

Chemical structures of tetracycline(A) and

streptomycine(B). (H.B.Wan,J.Liu,Talanta,45,1998,663)
3/4/2023
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MK. ANALISIS ELEKTROKIMIA

THEORY OF ELECTROPHORESIS APLIKASI ELEKTROFORESIS GEL

f: frictional coefficient, which depend on the Protein identification  Joule Heating

MK. ANALISIS ELEKTROKIMIA MK. ANALISIS ELEKTROKIMIA

MIGRASI SAMPEL ELEKTROFORESIS DAN BUFFER

IN MOST ELECTROPHORESIS UNITS, THE GEL IS MOUNTED BETWEEN TWO BUFFER

SLAB GEL UNIT FLAT BED UNIT MK. ANALISIS ELEKTROKIMIA

CONTINUOUS AND DISCONTINUOUS

MK. ANALISIS ELEKTROKIMIA

PROSES PEMISAHAN GEL INTERRELATION OF RESISTANCE,

 This can also be expressed as P = I2R

 In electrophoresis, one electrical parameter, either current,

THE NET CHARGE IS DETERMINED

TEMPERATURE AND ELECTROPHORESIS WHAT IS THE ROLE OF THE SOLID

AGAROSE AND POLYACRYLAMIDE AGAROSE AND POLYACRYLAMIDE

AGAROSE GELS STRUCTURE OF THE REPEATING UNIT OF

 Agarose dissolves when added to boiling liquid. It remains in a liquid

they are held together by the formation of weak hydrogen and

MK. ANALISIS ELEKTROKIMIA

 Polyacrylamide gels are tougher than agarose gels

 Acrylamide monomers polymerize into long chains that are

 Polyacrylamide is chemically complex, as is the production

MK. ANALISIS ELEKTROKIMIA

CROSSLINKING ACRYLAMIDE CHAINS CONSIDERATIONS WITH PAGE

Preparing and Pouring Gels

 Vary amount of crosslinker

Remove oxygen from mixture

MK. ANALISIS ELEKTROKIMIA  Photochemical method MK. ANALISIS ELEKTROKIMIA

CONSIDERATIONS WITH PAGE SDS GEL ELECTROPHORESIS

 Analysis of Gel  In SDS separations, migration is determined not by intrinsic electrical

MK. ANALISIS ELEKTROKIMIA

SDS GEL ELECTROPHORESIS

 A continuous system has only a single separating gel and uses

 In a discontinuous system a nonrestrictive large pore gel, called a

 The resolution obtainable in a discontinuous system is much

DETERMINING MOLECULAR WEIGHTS

 Run a gel with standard proteins of known

MK. ANALISIS ELEKTROKIMIA

 When a protein reaches its isoelectric point (pI) in the pH gradient,

TISSUE DISTRIBUTION OF MESSENGER A PROTEIN CAN BE SPECIFICALLY RECOGNIZED

GEL ELECTROPHORESIS OF DNA

Cathode (- Agarose slab gel

• Agarose is a polysaccharide derived from seaweed, which forms a

GEL ELECTROPHORESIS OF DNA GEL ELECTROPHORESIS OF DNA

PROCEDURES OF DNA FINGERPRINTING

MK. ANALISIS ELEKTROKIMIA MK. ANALISIS ELEKTROKIMIA

DNA POLYMORPHISMS DETECTION OF POLYMORPHISM

APPLICATION OF DNA FINGERPRINTING MUTATION HIGHLY VARIABLE REGIONS

• These regions are called hypervariable

MK. ANALISIS ELEKTROKIMIA MK. ANALISIS ELEKTROKIMIA

APPLICATION OF DNA FINGERPRINTING FORENSIC

MK. ANALISIS ELEKTROKIMIA MK. ANALISIS ELEKTROKIMIA

MK. ANALISIS ELEKTROKIMIA MK. ANALISIS ELEKTROKIMIA

AGAROSE GEL ELECTROPHORESIS

MK. ANALISIS ELEKTROKIMIA

Stain Detection limit Comment

Coomassie Blue 1.5 mg Permanent; high background

India Ink 100 ng Permanent

Silver stain 10 ng Permanent

Colloidal gold 3 ng Permanent

MK. ANALISIS ELEKTROKIMIA

 Senyawa mempunyai gugus planar yang dapat ber