EKTRAKSI DAN ISOLASI SENYAWA FITOKIMIA

Dr. RURINI RETNOWATI

FITOKIMIA
Phytochemical = kimia tumbuhan
Fitokimia : (dalam
arti luas ) adalah segala jenis zat kimia atau nutrien yang diturunkan dari sumber tumbuhan, termasuk sayuran dan buah-buahan

Dalam penggunaan fitokimia memiliki definisi yang lebih sempit. Fitokimia biasanya digunakan untuk merujuk pada senyawa yang ditemukan pada tumbuhan yang tidak dibutuhkan untuk fungsi normal tubuh, tapi memiliki efek yang menguntungkan bagi kesehatan atau memiliki peran aktif bagi pencegahan penyakit

APA YANG DIMAKSUD DENGAN FITOKIMIA ?

Phytochemicals
Disebut juga Phytoprotectants Plant bioactive compounds

Phytochemicals
Compounds in plants not recognised as nutrients (ie. Not essential dietary factors such as vitamins, minerals, amino acids and fatty

acids)
No deficiency syndromes May affect mammalian biological functions

+/- Health

What do they do in plants?

structure pollination colour to stems, leaves, flowers & fruits growth & development of the plant inhibition of the growth of competing plants pathogen and predator resistance

Remember, they evolved to benefit the plant, not us!

TOXIC

Main types of phytochemicals

Terpenoids
Alkaloids Sulphur compounds

Phenolics/polyphenols

Terpenoids
Carotenoids

(25,000)
Plant sterols

active ingredients in essential oils (e.g. in herbs and spices)

powerful insect anti-feedants or attractants

carotenoids essential in photosynthesis protect from UV damage orange, red and yellow colours sources include tomatoes, peas, citrus fruits, carrots

Alkaloids

(12,000)

discourage attack by fungi, herbivores and pathogens toxic substances, naturally present in plants, including food plants, e.g. solanine in green and sprouting potatoes.

basis of many modern day prescription drugs e.g. codeine, morphine, atropine; also of heroin and cocaine.

historic use as poisons

food sources include coffee, chilli, contaminated rye lysergic acid

capsaicin

caffeine

Sulphur compounds

(1,000s)

(e.g. glucosinolates found in Brassicas, and derivatives of the sulphur amino acid cysteine, found in the onion family) Function uncertain Unpleasant taste to discourage grazers? Breakdown products give hot flavour to mustard and horseradish

Phenolics/polyphenols (8,000)
Skeletal function UV protection

Antioxidant function
Pollination Astringency

Immune system
Colouration (reds, purples, blues)

Examples of well-known phenolics Salicylic acid

Phytoestrogens

SEL TUMBUHAN
DINDING SEL
CH2OH OH O O OH O OH
1 1

CH2OH O O CH2OH OH O O CH2OH OH O O OH

1

GLIKOSIDA

OH OH

OH

dirajang halus Gerus/blender Putus ikatan glikosida

MEMBRAN PLASMA
Semi permiabel Lipid, protein,

Perbedaan tekanan osmosis luar dan dalam sel, sel pecah

Metanol ; air ; eter Pelarut dengan mol kecil Penetrasi kedalam sel

SITOPLASMA (MATRIKS) - TEMPAT PROSES MET. PRIM DAN SEK - METABOLIT TERLARUT DALAM PELARUT

METABOLIT SEKUNDER

SURVAI LAPANGAN
KOLEKSI TUMBUHAN SAMPEL

SPESIMEN HERBARIUM

SKRINING FITO KIMIA

LABORATORIUM

SKRINING FITOKIMIA
IDENTIFIKASI AWAL SENY. MET. SEKUNDER

a. Alkaloida
Metoda Culvenor Fitzgerald, 4 gram sampel dipotong halus, digerus dalam lumpang dengan bantuan pasir yang bersih, dibasahi dengan 10 ml kloroform, ditambah dengan kloroform amoniak 0,05 M, digerus kembali dan disaring kedalam tabung reaksi, tambahkan 0,5 ml asam sulfat 2 N, kocok dan biarkan terjadinya 2 lapisan. Ambil lapisan asam sulfat dan masukkan kedalam tabung reaksi dan kemudian tambahkan 1 tetes pereaksi Mayer.

Terbentuknya endapan putih, positif alkaloid.

b. Terpenoida, Steroida, fenolik, flavonoida dan Saponin.

4 gram sampel segar dirajang halus didihkan dengan 25 mL etanol selama lebih kurang 25 menit disaring dalam keadaan panas, kemudian pelarut diuapkan sampai kering.

Ekstrak dikocok kuat dengan kloroforom lalu ditambahkan air suling.

Biarkan sampai terbentuk dua lapisan .

1.

Lapisan kloroform

diteteskan pada pelat tetes dan biarkan kering, tambahkan beberapa tetes asam asetat anhidrat dan asam sulfat pekat (pereaksi Libermann - Burchard). Terbentuknya warna :

merah,atau pink atau violet; (+)utk terpenoida. biru atau hijau ;(+) untuk steroida

Lapisan air :
Ambil 1 mL, dikocok selama 1 menit terbentuknya busa yang tidak hilang selama 5 menit, menandakan adanya saponin. Beberapa tetes ditempatkan dalam tabung reaksi di tambahkan besi(III) klorida, timbul warna hijau sampai ungu menandakan adanya fenolik Beberapa tetes ditempatkan dalam tabung reaksi, ditam bahkan asam khlorida pekat dan serbuk magnesium dan timbulnya warna merah positif flavonoida.

PEREAKSI BASA NITROGEN (ALKALOID) 1. MAYER, - 5 gr. KI , dilarutkan dalam 90 ml. Air, - tambahkan perlahan-lahan HgCl2 sambil diaduk. - Volume larutan dijadikan 100 ml. 2. DRAGENDORF

larutan a,

larutkan 16 g. KI dalam 40 ml. Air. larutkan 0,85 g. Bismut nitrat dan 10 g. asam tartarat dalam 40 ml. air Campurkan, lar a dan lar b (1:1) v/v, dan simpan pada suhu 0 o C Penggunaan, ambil 5 ml campuran dan larutkan dalam 50 ml air.

Larutan b

EKSTRAKSI :

MEMPEROLEH EKSTRAK

EKSTRAK : HASIL PREPARASI BERUPA CRUDE YANG MENGANDUNG TOTAL SENYAWA PENYUSUN SAMPEL TANAMAN YANG LARUT DALAM PELARUT TERTENTU

In dry extracts all solvent has been removed. Soft extracts and fluid extracts are prepared with mixtures of water and ethanol as solvent. Tinctures are prepared by extraction of the crude drug with five to ten parts of ethanol of varying concentration, without concentration of the final product.

PROSEDUR EKSTRAKSI

BEBERAPA CARA UNTUK MEMPEROLEH EKSTRAK:

1. 2. 3. 4. 5. 6.

7.
8.

Infusion Maceration Percolation Digestion Decoction Continuous hot extraction Liquid-liquid extraction Chromatography

EKTRAKSI TANAMAN
Pemilihan prosedur ekstraksi tergantung pada : - Sifat material tanaman - Komponen senyawa yang akan diisolasi - Sampel kering sebaiknya dibuat dalam sediaan serbuk sebelum di ekstraksi - Sampel segar (daun, bunga) dihomogenkan dengan pelarut (alkohol) - Alkohol juga digunakan untuk menstabilkan daun segar.

ISOLASI/PEMISAHAN DAN PEMURNIAN

MEMPEROLEH : ISOLAT FRAKSI SENYAWA MURNI /MOLEKUL TUNGGAL

ISOLASI DAN PEMISAHAN SENYAWA PENYUSUN

Proses isolasi dan pemisahan senyawa merupakan tahapan yang paling sulit dalam penelitian fitokima

INFUDASI

INFUDASI :
METODE EKSTRAKSI SENYAWA KIMIA ATAU BAGIAN TANAMAN ( BATANG, AKAR, KULIT KAYU, RIMPANG) DENGAN CARA MENDIDIHKANNYA DALAM AIR SELAMA 15 MENIT

PROSEDUR :

BAGIAN TANAMAN DIHANCURKAN DAN DIBUAT BUBUK, KEMUDIAN DITAMBAHKAN AIR DAN DIDIDIHKAN SELAMA 15 MENIT. PROSES INI AKAN MELARUTKAN MINYAK, SENYAWA ORGANIK VOLATIL DAN BEBERAPA SENYAWA LAINNYA.
CONTOH : MEMBUAT ADONAN TEH

DEKOKTASI

DEKOKTASI :
METODE EKSTRAKSI SENYAWA KIMIA ATAU BAGIAN TANAMAN ( BATANG, AKAR, KULIT KAYU, RIMPANG) DENGAN CARA MENDIDIHKANNYA DALAM AIR SELAMA 25 30 MENIT

PROSEDUR :

BAGIAN TANAMAN DIHANCURKAN DAN DIBUAT BUBUK, KEMUDIAN DITAMBAHKAN AIR DAN DIDIDIHKAN SELAMA 30 MENIT. PROSES INI AKAN MELARUTKAN MINYAK, SENYAWA ORGANIK VOLATIL DAN BEBERAPA SENYAWA LAINNYA.
CONTOH : MEMBUAT BLACK COFFEE

MASERASI
MASERASI (PERENDAMAN ) ; TEKNIK MASERASI DIGUNAKAN JIKA SENYAWA METABOLIT SEKUNDER DALAM TANAMAN CUKUP BESAR PROSENTASENYA DAN DITEMUKAN PELARUT YANG DAPAT MELARUTKAN SENYAWA TERSEBUT TANPA PEMANASAN. CARA INI MEMBUTUHKAN WAKTU LAMA DAN PELARUT DALAM JUMLAH BESAR, SERTA SULIT MENCARI PELARUT YANG MELARUTKAN DENGAN BAIK SEMUA SENYAWA. APABILA GOLONGAN SENYAWA YANG AKAN DIISOLASI SUDAH DIKETAHUI CARA INI BAIK DIGUNAKAN

MASERASI

PERKOLASI
TEKNIK PERKOLASI HAMPIR SAMA DENGAN MASERASI. TEKNIK PERKOLASI MERUPAKAN METODE EKSTRAKSI DENGAN PELARUT, DIMANA PELARUT TERSEBUT DILEWATKAN SECARA PERLAHAN (TETES DEMI TETES).

BIASANYA DIGUNAKAN PELARUT YANG TIDAK MUDAH MENGUAP TETAPI MELARUTKAN SENYAWA DALAM TANAMAN DENGAN BAIK. DAN JANGAN GUNAKAN PELARUT YANG BERBAU KERAS
DIGUNAKAN APABILA PRESENTASE SENYAWA CUKUP BESAR.

EKSTRAKSI SOXHLET
EKSTRAKSI KONTINUE

ALAT SOXHLET

KONSEP
EKSTRAKSI PERUBAHAN KEADAAN/WUJUD KELARUTAN

Extractions are used when we want to separate substances. One way this can be done is by using a solvent in which a desired substance dissolves in and the undesired substance does not dissolve in.

LIKE DISSOLVE LIKE

PERUBAHAN WUJUD (STATE)

EVAPORASI
-

Fenomena evaporasi : dimana suatu molekul cair mempunyai energi yang cukup untuk berubah ke fase gas tanpa pemanasan/pendidihan Evaporasi dapat terjadi pada temperatur yang berdekatan, pada keadaan tertentu suatu molekul cair mempunyai energi yang cukup untuk lepas ke udara.
Fenomena kondensasi : dimana suatu molekul gas kehilangan energi untuk beruwujud gas dan di koleksi pada permukaan yang dingin sebagai cairan

Condensation
-

KELARUTAN Merupakan fungsi kelarutan suatu senyawa dalam suatu pelarut tertentu

PELARUT YANG DIGUNAKAN UNTUK EKSTRAKSI TANAMAN

Alkohol merupakan pelarut yang umum digunakan. Selain itu pelarut yang tidak bercampur dengan air , misal : petroleum eter fraksi rendah ( volatil, minyak, steroid), eter, kloroform (alkaloid, kuinon).

EXTRACTION OF ALKALOIDS & PHENOLS

Before extracting alkaloids (organic bases), basification of the plant material is necessary (if a water immiscible solvent is used). If aromatic acids & phenols are going to be extracted, acidification may be required.

SUBLIMATION
Sublimation is sometimes possible on whole drugs (e.g. isolating caffeine from tea). Modern equipment uses low pressures with a strict control of temperature.

DISTILLATION
i. FRACTIONAL DISTILLATION: traditional method of separation of constituents of volatile mixtures (isolation of components of volatile oils). ii. STEAM DISTILLATION: used to isolate volatile oils and hydrocyanic acid from plant material.

KROMATOGRAFI
METODE PEMISAHAN SUATU CAMPURAN BERDASARKAN PERBEDAAN DISTRIBUSINYA DALAM FASA DIAM (STATIONARY PHASE) DAN FASA GERAK (MOBILE PHASE) Chromatography (from Greek :chroma, color and :graphein to write) .

FASA DIAM : CAIR, PADAT FASA GERAK : GAS, CAIR KROMATOGRAFI PEMISAHAN DAN PEMURNIAN

ADSORPTION CHROMATOGRAPHY
In its simplest form, this method of chromatography consists of passing a solution of the mixture of compounds needing to be separated, through a hollow glass column, packed with a finely divided absorbent powder, and collecting the solution (eluate).

The surface phenomenon of adsorption is utilized. The finely-divided solids are capable of selective adsorption of other substances. The components of a mixture introduced onto a column of adsorbent are more or less strong adsorbed. Those which are least strongly adsorbed are carried down the column by the passage of solvent, & are the 1st to be eluted from the bottom of the column.

TAHAPAN PEMISAHAN/PEMURNIAN SENYAWA

KROMATOGRAFI KOLOM Eluen kepolaran tetap ~ kepolaran bertingkat

TLC
KUMPULKAN FRAKSI DENGAN Rf YANG SAMA

uapkan pelarut
ROTARY EVAPORATOR
FRAKSI-FRAKSI

OILY/CAIR KRISTAL PADATAN AMORF

KROMATOGRAFI KOLOM
FASA DIAM :
SILICA GEL, ALUMINA, CELLULOSE

FASA GERAK :

(ELUEN0 -PELARUT DENGAN BERBAGAI KOMPOSISI -DIPILIH DARI HASIL KLT (TLC)

TAHAPAN PROSES KROMATOGRAFI KOLOM

FASA DIAM : PADATAN FASA GERAK : CAIRAN

THIN LAYER CHROMATOGRAPHY (TLC)

TLC is an e.g. of adsorption chromatography, the stationary phase being a thin layer adsorbent held on a suitable backing. Separation of the compounds present in the plant extract depends on the differences in their adsorptive/desorptive behaviour in respect of the stationary phase.

TLC involves a thin layer of adsorbent, mixed with a binder such as CaSo4, which is spread on a glass plate & allowed to dry. The plant mixture to be separated is applied as a spot near the base of the plate, which is then placed in a closed glass tank containing a a layer of developing solvent.

The solvent moves up the plate by capillary action, carrying with it the less strongly adsorbed components of the mixture, while the more strongly adsorbed compounds remain near the base of the plate. When the solvent has reached 1-2 cm from the top of the plate, the plate is removed from the tank & dried.

The now separated components of the mixture appear as spots on the finished plate (chromatogram), corresponding to the bands of the adsorbent column.

TLC THE ADSORBENT

With adsorption TLC, different substances have different adsorptive capacities & any one material can vary in its activity according to the pre-treatment of the TLC plate. The absorbent must be chosen in relation to the properties of the solvent & the mixture to be separated. In general: if a highly active adsorbent is used, then a solvent with a corresponding high power of elution for this substance will be required. E.g. Aluminium (acidic, neutral or basic) with different activity grades is commonly used.

TLC THE SOLVENT

Solvents used for TLC must be pure. Commonly used solvents - Methanol - Ethanol - And other alcohols - Chloroform - Ether - Ethyl acetate

VISIBILITY OF SPOTS (COMPOUNDS)

If invisible, the spots may be made visible by - Heating for a specific period - Examining under U.V light (if substances are florescent). - Spraying the finished chromatogram with a suitable reagent e.g. iodine & Dragendorffs reagent are used as sprays for the general detection of iodine (although they are not specific for alkaloids).

ADVANTAGES OF TLC
-

Simple, inexpensive Quick results can be achieved from between 30 minutes to a few hours Good separation of spots (compounds) Very sensitive The chromatogram is resistant to the action of chemicals used for the visualization of compounds.

COMPONENTS OF THE TLC SYSTEM

3 components
i. ii. iii.

THE ADSORBENT Stationary Phase THE ELUENT (THE DEVELOPING SOLVENT) Mobile Phase THE SUBSTANCE REQUIRING SEPARATION Plant Sample

In practice, only 2 adsorbents are widely used - Silica gel - Aluminium When using these adsorbents, the rule is to match the polarity of the developing solvent with that of the compounds of the mixture to be chromatographed.

SEPARATION OF ALKALOIDS
Alkaloids need a moderately polar solvent for good separation (e.g. ether/ethanol: 95/5).
A more polar solvent (e.g. pure methanol), would be preferentially adsorbed, & the alkaloids would be carried along by the passage of the solvent resulting in poor separation. On the other hand, a non-polar solvent (e.g. cyclohexane) would be unable to displace the alkaloids from the adsorbent layer & they would then remain at or near the base of origin.

ADDITIONAL FACTOR FOR SEPARATING ALKALOIDS

If using an aluminium thin layer (neutral), a neutral solvent should be used. If using Si-gel (slightly acidic due to the method of preparation), and alkaline solvent such as acetone/water/25%am monia: 90/7/3 makes for good separation.

SEPARATION OF VOLATILE OILS

The constituents of volatile oils are mainly non-polar (terpenes) & are therefore best separated with corresponding nonpolar solvents such as chloroform/benzene mixtures. Certain oils may need more polar solvents (e.g. clove oil phenolic).

SEPARATION OF SUGARS & SUGAR ACID MIXTURES

These mixtures are produced by hydrolysing starch & gums. They are strongly polar & are therefore strongly adsorbed onto silica & aluminium layers.

GENERAL RULE FOR TLC

TLC is best used for moderately or weakly polar mixtures.

PARTITION CHROMATOGRAPHY
Partition chromatography is based on the differences in partition co-efficients of the compounds of a mixture which have to be separated, between an aqueous & immiscible organic liquid. The stationary phase is normally aqueous, which is mixed with an inert carrier powder & packed into a glass column.

PARTITION CHROMATOGRAPHY ON PAPER

Although it has been for a large part been replaced by TLC, it still remains the method of choice for the separation of some types of compounds.

METHOD OF PAPER CHROMATOGRAPHY

The solution to be separated is applied as a spot near one end of a prepared filter-paper strip The paper is then supported in an airtight chamber which has an atmosphere saturated with solvent & water, and a supply of the watersaturated solvent.

The best solvents are those which are partially miscible with water (phenol, n-butanol & amyl alcohol).
Either the paper may be dipped in the solvent mixture so that the solvent travels up the paper (ascending technique) or a trough of solvent may be supported at the top of the chamber so it travels down the paper (descending technique).

As the solvent moves, the compounds also move along the paper at varying rates, depending on the differences in their partition coefficients between the aqueous & organic phases.

VISIBILITY OF COMPOUNDS
After the filter-paper strips have been dried, the positions of the separated components can be revealed by the use of suitable developing agents: - Ninhydrin solution amino acids - Iodine solution/vapour or modified Dragendorffs reagent alkaloids - Ferric chloride solution phenols - Alkali anthraquinones - Antimony trichloride in chloroform steroids & some volatile oil components - Aniline hydrogen phthalate reagent - sugars

SEPARATION OF SUBSTANCES
For the separation of substances, it is necessary to use a 2-D chromatogram.
First one solvent is run in one direction, then, after drying of the paper, a 2nd solvent is run in a direction at right angles to the 1st This is especially applicable to mixtures of amino acids.

PAPER CHROMATOGRAPHY
The ratio between the distance travelled on the paper by a component of the test solution & the distance travelled by the solvent is termed the RF value. Under standard conditions, this is a constant for the particular compound.

In practise, however, variations of the RF value often occur & it is best to run a reference compound alongside the unknown mixtures.

PAPER CHROMATOGRAPHY: ADVANTAGES & DISADVANTAGES

ADVANTAGES i. Simple & inexpensive ii. Sensitive gives good separation of very small amounts, of especially water-soluble compounds, e.g. sugars.
DISADVANTAGES i. Fragile chromatogram may be destroyed by chemicals used for visualization ii. May be time-consuming.

RF VALUES
Rf (rate of flow) values are used as a way of identifying compounds on a chromatogram. In theory, the Rf value is constant in a constant set of chromatographic conditions. In practice, it is difficult to achieve because of the many factors that may affect the Rf value.

RF VALUES & MOISTURE

Very NB:= amount of moisture adsorbed on the thin layer. The presence of significant amounts of moisture decrease the number of sites available for active adsorption. The compounds undergoing separation will therefore have a higher than normal Rf value. The moisture content of thin layer plates is difficult to control & is therefore a major factor in nonreproducibility of Rf values.

RF & TANK SATURATION

In a non-saturated tank, the solvent evaporates as it travels up the plate producing a lower solvent front than in a saturated tank. Rf values of substances being separated will therefore be higher than these obtained using a saturated tank.

Developing the Plates

After preparing the development chamber and spotting the samples, the plates are ready for development. Be careful to handle the plates only by their edges, and try to leave the development chamber uncovered for as little time as possible.

When the plates are removed from the chamber, quickly trace the solvent front (the highest solvent level on the plate) with a pencil.

Identifying the Spots

If the spots can be seen, outline them with a pencil. If no spots are obvious, the most common visualization technique is to hold the plate under a UV lamp (CAUTION: Do not look directly into the lamp.) Many organic compounds can be seen using this technique, and many commercially made plates often contain a substance which aids in the visualization of compounds.

Identifying the Spots

Commercial TLC plate after development in normal lighting

Same TLC plate held under a UV lamp Note the appearance of additional spots

Interpreting the Data

The Rf value for each spot should be calculated. Rf stands for "ratio of fronts" and is characteristic for any given compound on the same stationary phase using the same mobile phase for development of the plates. Hence, known Rf values can be compared to those of unknown substances to aid in their identifications.

Interpreting the Data

(Note: Rf values often depend on the temperature and the solvent used in the TLC experiment; the most effective way to identify a compound is to spot known substances next to unknown substances on the same plate). In addition, the purity of a sample may be estimated from the chromatogram. An impure sample will often develop as two or more spots, while a pure sample will show only one spot.

RECAP - PC
Paper chromatography is one method for testing the purity of compounds and identifying substances. Paper chromatography is a useful technique because it is relatively quick and requires small quantities of material.

PC - DESCRIPTION
Separations in paper chromatography involve the same principles as those in thin layer chromatography. In paper chromatography, like thin layer chromatography, substances are distributed between a stationary phase and a mobile phase. The stationary phase is usually a piece of high quality filter paper. The mobile phase is a developing solution that travels up the stationary phase, carrying the samples with it. Components of the sample will separate on the stationary phase according to how strongly they adsorb to the stationary phase versus how much they dissolve in the mobile phase.

Preparing the Chamber

Choose a developing chamber that can be sealed well. The chamber should be large enough to hold the paper that is to be developed. The chamber should be clean and dry before use. Add the mobile phase to the chamber so that it is about 2 cm deep. Seal the chamber tightly and let the chamber stand overnight if possible (Allowing the chamber to stand permits its atmosphere to become saturated with the developing solvent. A saturated atmosphere allows for more effective development of the chromatograms. The larger the chamber, the longer it should stand )

Preparing the Chamber

The larger the chamber, the longer it should stand

Preparing the Stationary Phase

Cut a square piece of high-quality filter paper to fit into your development chamber. With a pencil, draw a straight line about 3 cm from the bottom edge of the paper.

Spotting the Samples

First, each sample should be dissolved in an appropriate solvent to make about a one percent solution (0.01 g sample/1 g solvent). Less than one milliliter of solution will be needed for the experiment. Then the dissolved samples may be spotted to the paper.

Spotting the Samples

If a larger quantity of sample is needed for the experiment than is provided by one application, the solution may be re-spotted. All spots on the chromatogram should be 2 to 2.5 cm away from the edges of the paper and from each other.

Developing the Chromatograms

After preparing the chamber and spotting the samples, the paper is ready for development. Be careful to handle the paper only by its edges, and try to leave the development chamber uncovered for as little time as possible. Initially, the chromatogram should be suspended in the chamber without touching the solvent. To suspend the chromatogram, to the top of the paper and thread a piece of string throught the paper clip. Then tape the string to the outside of the chamber to hold the chromatogram in place. The paper should hang in the development chamber overnight, if possible.

Developing the Chromatograms

After the chromatogram has hung in the chamber, immerse the paper's bottom edge into the developing solvent. Allow the chromatogram to dry in a well-ventilated area.

Identifying the Spots

If the spots can be seen, outline them with a pencil. If the spots are not obvious, the most common visualization technique is to hold the paper under an ultraviolet lamp. (Caution: Do not look directly into the lamp!) Many organic compounds can be seen using this technique. Outline the spots with a pencil.

PERALATAN KROMATOGRAFI GAS

KROMATOGRAM MINYAK NILAM DENGAN KOLOM PACKING DAN KOLOM KAPILER

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) HIGH PRESSURE LIQUID CHROMATOGRAPHY is a form of column chromatography used frequently in biochemistry and analytical chemistry to separate, identify, and quantify compounds. HPLC utilizes a column that holds chromatographic packing material (stationary phase), a pump that moves the mobile phase(s) through the column, and a detector that shows the retention times of the molecules. Retention time varies depending on the interactions between the stationary phase, the molecules being analyzed, and the solvent(s) used.

FASA DIAM : PADATAN FASA GERAK : CAIRAN DIGUNAKAN UNTUK MEMISAHKAN DAN MENGANALISIS CAMPURAN SENYAWA YANG BERSIFAT KURANG VOLATIL

PERALATAN HPLC