EKTRAKSI DAN ISOLASIEKTRAKSI DAN ISOLASISENYAWA FITOKIMIASENYAWA FITOKIMIA

Dr. RURINI RETNOWATI

FITOKIMIAPhytochemical = 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 (25,000)

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

Carotenoids Plant sterols

caffeine

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

capsaicin lysergic acid

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

O

CH2OH

OH

OH

1

1

OH

OH

OH

CH2OH

O

1

OH

OH

OH

OH

O

1

OH

OH

OH

OH

CH2OH

O

1

OH

OH

CH2OH

O

O

O

O

O

O

Perbedaan tekanan osmosis luar dan dalam sel, sel

pecah

dirajang halus Gerus/blenderPutus ikatan glikosida

MEMBRAN PLASMASemi permiabelLipid, protein,

Metanol ; air ; eterPelarut dengan mol kecilPenetrasi kedalam sel

SEL TUMBUHAN

DINDING SEL GLIKOSIDA

SITOPLASMA (MATRIKS)

-   TEMPAT PROSES MET. PRIM DAN SEK-   METABOLIT TERLARUT DALAM PELARUT

METABOLIT SEKUNDER

SPESIMEN

HERBARIUM

SAMPEL

KOLEKSI TUMBUHAN

LABORATORIUM

SURVAI LAPANGAN

SKRININGFITO KIMIA

IDENTIFIKASI AWAL SENY. MET. SEKUNDER

a. Alkaloidaa. Alkaloida

Metoda Culvenor Fitzgerald,Metoda Culvenor Fitzgerald,   

4 gram sampel dipotong halus, digerus dalam lumpang 4 gram sampel dipotong halus, digerus dalam lumpang

dengan dengan

bantuan pasir yang bersih, dibasahi dengan 10 ml kloroform, bantuan pasir yang bersih, dibasahi dengan 10 ml kloroform,

ditambah dengan kloroform amoniak 0,05 M, ditambah dengan kloroform amoniak 0,05 M,

digerus kembali dan disaring kedalam tabung reaksi, digerus kembali dan disaring kedalam tabung reaksi,

tambahkan 0,5 ml asam sulfat 2 N, kocok dan biarkan tambahkan 0,5 ml asam sulfat 2 N, kocok dan biarkan

terjadinya terjadinya

2 lapisan. Ambil lapisan asam sulfat dan masukkan kedalam2 lapisan. Ambil lapisan asam sulfat dan masukkan kedalam

tabung reaksi dan kemudian tambahkan 1 tetes pereaksitabung reaksi dan kemudian tambahkan 1 tetes pereaksi

Mayer.Mayer.

Terbentuknya endapan putih, positif alkaloid.Terbentuknya endapan putih, positif alkaloid.

SKRINING FITOKIMIASKRINING FITOKIMIA

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)PEREAKSI BASA NITROGEN (ALKALOID)

1. MAYER, 1. MAYER, - 5 gr. KI , dilarutkan dalam 90 ml. Air,- 5 gr. KI , dilarutkan dalam 90 ml. Air, - tambahkan perlahan-lahan HgCl2 sambil diaduk. - tambahkan perlahan-lahan HgCl2 sambil diaduk. - Volume larutan dijadikan 100 ml.- Volume larutan dijadikan 100 ml.

2. DRAGENDORF2. DRAGENDORF larutan alarutan a, , larutkan 16 g. KI dalam 40 ml. Air.larutkan 16 g. KI dalam 40 ml. Air.   Larutan bLarutan b larutkan 0,85 g. Bismut nitrat dan 10 g. asam tartarat larutkan 0,85 g. Bismut nitrat dan 10 g. asam tartarat dalam 40 ml. airdalam 40 ml. air   Campurkan,Campurkan, lar a dan lar b (1:1) v/v, dan simpan pada suhu 0 o Clar a dan lar b (1:1) v/v, dan simpan pada suhu 0 o C Penggunaan, Penggunaan, ambil 5 ml campuran dan larutkan dalam 50 ml air.ambil 5 ml campuran dan larutkan dalam 50 ml air.

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.

BEBERAPA CARA UNTUK MEMPEROLEH EKSTRAK:1. Infusion √2. Maceration √3. Percolation √4. Digestion5. Decoction √6. Continuous hot extraction √7. Liquid-liquid extraction8. Chromatography √

PROSEDUR EKSTRAKSI

EKTRAKSI TANAMANEKTRAKSI TANAMAN

Pemilihan prosedur ekstraksi Pemilihan prosedur ekstraksi tergantung pada :tergantung pada :

- Sifat material tanaman- Sifat material tanaman-Komponen senyawa yang akan Komponen senyawa yang akan diisolasidiisolasi-Sampel kering sebaiknya dibuat Sampel kering sebaiknya dibuat dalam sediaan serbuk sebelum di dalam sediaan serbuk sebelum di ekstraksiekstraksi-Sampel segar (daun, bunga) Sampel segar (daun, bunga) dihomogenkan dengan pelarut dihomogenkan dengan pelarut (alkohol)(alkohol)-Alkohol juga digunakan untuk Alkohol juga digunakan untuk menstabilkan daun segar.menstabilkan daun segar.

ISOLASI/PEMISAHAN DAN PEMURNIAN

MEMPEROLEH : ISOLAT FRAKSI SENYAWA MURNI /MOLEKUL TUNGGAL

ISOLASI DAN PEMISAHAN ISOLASI DAN PEMISAHAN SENYAWA PENYUSUNSENYAWA PENYUSUN

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

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

DEKOKTASI

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

• 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.

PERKOLASI

EKSTRAKSI SOXHLETEKSTRAKSI 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.

Condensation- Fenomena kondensasi : dimana suatu molekul gas kehilangan energi untuk

beruwujud gas dan di koleksi pada permukaan yang dingin sebagai cairan

KELARUTANMerupakan fungsi kelarutan suatu senyawa dalam suatu pelarut tertentu

PELARUT YANG DIGUNAKAN PELARUT YANG DIGUNAKAN UNTUK EKSTRAKSI TANAMANUNTUK EKSTRAKSI TANAMAN

Alkohol merupakan Alkohol merupakan pelarut yang umum pelarut yang umum digunakan.digunakan.

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

EXTRACTION OF ALKALOIDS & EXTRACTION OF ALKALOIDS & PHENOLSPHENOLS

Before extracting Before extracting alkaloids (organic alkaloids (organic bases), basification of bases), basification of the plant material is the plant material is necessary (if a water necessary (if a water immiscible solvent is immiscible solvent is used).used).

If aromatic acids & If aromatic acids & phenols are going to phenols are going to be extracted, be extracted, acidification may be acidification may be required. required.

SUBLIMATIONSUBLIMATION

Sublimation is Sublimation is sometimes possible sometimes possible on whole drugs (e.g. on whole drugs (e.g. isolating caffeine from isolating caffeine from tea).tea).

Modern equipment uses Modern equipment uses low pressures with a low pressures with a strict control of strict control of temperature.temperature.

DISTILLATIONDISTILLATIONi. FRACTIONAL i. FRACTIONAL

DISTILLATION: traditional DISTILLATION: traditional method of separation of method of separation of constituents of volatile constituents of volatile mixtures (isolation of mixtures (isolation of components of volatile components of volatile oils).oils).

ii. STEAM DISTILLATION: ii. STEAM DISTILLATION: used to isolate volatile oils used to isolate volatile oils and hydrocyanic acid from and hydrocyanic acid from plant material.plant material.

KROMATOGRAFI

METODE PEMISAHAN SUATU CAMPURAN BERDASARKAN PERBEDAAN DISTRIBUSINYA DALAM FASA DIAM (STATIONARY PHASE) DAN FASA GERAK (MOBILE PHASE)

Chromatography (from Greek χρώμα:chroma, color and γραφειν:graphein to write) .

FASA DIAM : CAIR, PADATFASA GERAK : GAS, CAIR

KROMATOGRAFI PEMISAHAN DAN PEMURNIAN

ADSORPTION ADSORPTION CHROMATOGRAPHYCHROMATOGRAPHY

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

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

FRAKSI-FRAKSI

TLCKUMPULKAN FRAKSI

DENGAN Rf YANG SAMA

uapkan pelarut

ROTARY EVAPORATOR

OILY/CAIRKRISTALPADATAN AMORF

TAHAPAN PEMISAHAN/PEMURNIAN SENYAWA

KROMATOGRAFI KOLOMEluen kepolaran tetap ~ kepolaran bertingkat

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 : PADATANFASA GERAK : CAIRAN

THIN LAYER THIN LAYER CHROMATOGRAPHY (TLC)CHROMATOGRAPHY (TLC)

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

TLC involves a thin layer TLC involves a thin layer of adsorbent, mixed of adsorbent, mixed with a binder such as with a binder such as CaSo4, which is CaSo4, which is spread on a glass spread on a glass plate & allowed to dry.plate & allowed to dry.

The plant mixture to be The plant mixture to be separated is applied separated is applied as a spot near the as a spot near the base of the plate, base of the plate, which is then placed in which is then placed in a closed glass tank a closed glass tank containing a a layer of containing a a layer of developing solvent.developing solvent.

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

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

TLC – THE ADSORBENTTLC – THE ADSORBENT

With adsorption TLC, different substances have With adsorption TLC, different substances have different adsorptive capacities & any one different adsorptive capacities & any one material can vary in its activity according to the material can vary in its activity according to the pre-treatment of the TLC plate.pre-treatment of the TLC plate.

The absorbent must be chosen in relation to the The absorbent must be chosen in relation to the properties of the solvent & the mixture to be properties of the solvent & the mixture to be separated.separated.

In general: if a highly active adsorbent is used, In general: if a highly active adsorbent is used, then a solvent with a corresponding high power then a solvent with a corresponding high power of elution for this substance will be required.of elution for this substance will be required.

E.g. Aluminium (acidic, neutral or basic) with E.g. Aluminium (acidic, neutral or basic) with different activity grades is commonly used.different activity grades is commonly used.

TLC – THE SOLVENTTLC – THE SOLVENT

Solvents used for TLC must be pure.Solvents used for TLC must be pure.Commonly used solventsCommonly used solvents- MethanolMethanol- EthanolEthanol- And other alcoholsAnd other alcohols- ChloroformChloroform- EtherEther- Ethyl acetateEthyl acetate

VISIBILITY OF SPOTS VISIBILITY OF SPOTS (COMPOUNDS)(COMPOUNDS)

If invisible, the spots may be If invisible, the spots may be made visible bymade visible by

- Heating for a specific periodHeating for a specific period- Examining under U.V light (if Examining under U.V light (if

substances are florescent).substances are florescent).- Spraying the finished Spraying the finished

chromatogram with a suitable chromatogram with a suitable reagent e.g. iodine & reagent e.g. iodine & Dragendorff’s reagent are used Dragendorff’s reagent are used as sprays for the general as sprays for the general detection of iodine (although detection of iodine (although they are not specific for they are not specific for alkaloids).alkaloids).

ADVANTAGES OF TLCADVANTAGES OF TLC

- Simple, inexpensiveSimple, inexpensive- Quick – results can be achieved from Quick – results can be achieved from

between 30 minutes to a few hoursbetween 30 minutes to a few hours- Good separation of spots (compounds)Good separation of spots (compounds)- Very sensitiveVery sensitive- The chromatogram is resistant to the The chromatogram is resistant to the

action of chemicals used for the action of chemicals used for the visualization of compounds.visualization of compounds.

COMPONENTS OF THE TLC COMPONENTS OF THE TLC SYSTEMSYSTEM

3 components3 components

i.i. THE ADSORBENT – Stationary PhaseTHE ADSORBENT – Stationary Phase

ii.ii. THE ELUENT (THE DEVELOPING THE ELUENT (THE DEVELOPING SOLVENT) – Mobile PhaseSOLVENT) – Mobile Phase

iii.iii. THE SUBSTANCE REQUIRING THE SUBSTANCE REQUIRING SEPARATION – Plant SampleSEPARATION – Plant Sample

In practice, only 2 In practice, only 2 adsorbents are widely adsorbents are widely usedused

- Silica gelSilica gel- AluminiumAluminium

When using these When using these adsorbents, the rule is adsorbents, the rule is to match the polarity to match the polarity of the developing of the developing solvent with that of solvent with that of the compounds of the the compounds of the mixture to be mixture to be chromatographed.chromatographed.

SEPARATION OF ALKALOIDSSEPARATION OF ALKALOIDSAlkaloids need a moderately polar Alkaloids need a moderately polar

solvent for good separation (e.g. solvent for good separation (e.g. ether/ethanol: 95/5). ether/ethanol: 95/5).

A more polar solvent (e.g. pure A more polar solvent (e.g. pure methanol), would be preferentially methanol), would be preferentially adsorbed, & the alkaloids would be adsorbed, & the alkaloids would be carried along by the passage of carried along by the passage of the solvent the solvent resulting in poor resulting in poor separation. separation.

On the other hand, a non-polar On the other hand, a non-polar solvent (e.g. cyclohexane) would solvent (e.g. cyclohexane) would be unable to displace the alkaloids be unable to displace the alkaloids from the adsorbent layer & they from the adsorbent layer & they would then remain at or near the would then remain at or near the base of origin.base of origin.

ADDITIONAL FACTOR FOR ADDITIONAL FACTOR FOR SEPARATING ALKALOIDSSEPARATING ALKALOIDS

If using an aluminium thin If using an aluminium thin layer (neutral), a neutral layer (neutral), a neutral solvent should be used. solvent should be used.

If using Si-gel (slightly If using Si-gel (slightly acidic due to the acidic due to the method of preparation), method of preparation), and alkaline solvent and alkaline solvent such as such as acetone/water/25%amacetone/water/25%ammonia: 90/7/3 makes monia: 90/7/3 makes for good separation.for good separation.

SEPARATION OF VOLATILE OILSSEPARATION OF VOLATILE OILS

The constituents of volatile The constituents of volatile oils are mainly non-polar oils are mainly non-polar (terpenes) & are (terpenes) & are therefore best separated therefore best separated with corresponding non-with corresponding non-polar solvents such as polar solvents such as chloroform/benzene chloroform/benzene mixtures.mixtures.

Certain oils may need Certain oils may need more polar solvents (e.g. more polar solvents (e.g. clove oil – phenolic).clove oil – phenolic).

SEPARATION OF SUGARS & SEPARATION OF SUGARS & SUGAR ACID MIXTURESSUGAR ACID MIXTURES

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

GENERAL RULE FOR TLCGENERAL RULE FOR TLC

TLC is best used for TLC is best used for moderately or weakly moderately or weakly polar mixtures.polar mixtures.

PARTITION CHROMATOGRAPHYPARTITION CHROMATOGRAPHY

Partition chromatography is based on the Partition chromatography is based on the differences in partition co-efficients of the differences in partition co-efficients of the compounds of a mixture which have to be compounds of a mixture which have to be separated, between an aqueous & separated, between an aqueous & immiscible organic liquid.immiscible organic liquid.

The stationary phase is normally aqueous, The stationary phase is normally aqueous, which is mixed with an inert carrier powder which is mixed with an inert carrier powder & packed into a glass column.& packed into a glass column.

PARTITION CHROMATOGRAPHY PARTITION CHROMATOGRAPHY ON PAPERON PAPER

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

METHOD OF PAPER METHOD OF PAPER CHROMATOGRAPHYCHROMATOGRAPHY

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

The best solvents are those which are partially The best solvents are those which are partially miscible with water (phenol, n-butanol & amyl miscible with water (phenol, n-butanol & amyl alcohol). alcohol).

Either the paper may be dipped in the solvent Either the paper may be dipped in the solvent mixture so that the solvent travels up the mixture so that the solvent travels up the paper (ascending technique) or a trough of paper (ascending technique) or a trough of solvent may be supported at the top of the solvent may be supported at the top of the chamber so it travels down the paper chamber so it travels down the paper (descending technique).(descending technique).

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

VISIBILITY OF COMPOUNDSVISIBILITY OF COMPOUNDS

After the filter-paper strips have been dried, the After the filter-paper strips have been dried, the positions of the separated components can be positions of the separated components can be revealed by the use of suitable developing revealed by the use of suitable developing agents: agents:

- Ninhydrin solution – amino acids- Ninhydrin solution – amino acids- Iodine solution/vapour or modified Dragendorff’s - Iodine solution/vapour or modified Dragendorff’s

reagent – alkaloidsreagent – alkaloids- Ferric chloride solution – phenols- Ferric chloride solution – phenols- Alkali – anthraquinones- Alkali – anthraquinones- Antimony trichloride in chloroform – steroids & - Antimony trichloride in chloroform – steroids &

some volatile oil componentssome volatile oil components- Aniline hydrogen phthalate reagent - sugars- Aniline hydrogen phthalate reagent - sugars

SEPARATION OF SUBSTANCESSEPARATION OF SUBSTANCESFor the separation of For the separation of

substances, it is substances, it is necessary to use a 2-D necessary to use a 2-D chromatogram.chromatogram.

First one solvent is run in First one solvent is run in one direction, then, one direction, then, after drying of the after drying of the paper, a 2paper, a 2ndnd solvent is solvent is run in a direction at run in a direction at right angles to the 1right angles to the 1stst This is especially This is especially applicable to mixtures applicable to mixtures of amino acids.of amino acids.

PAPER CHROMATOGRAPHYPAPER CHROMATOGRAPHY

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

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

PAPER CHROMATOGRAPHY: PAPER CHROMATOGRAPHY: ADVANTAGES & DISADVANTAGESADVANTAGES & DISADVANTAGES

ADVANTAGESADVANTAGESi.i. Simple & inexpensiveSimple & inexpensiveii.ii. Sensitive – gives good separation of very Sensitive – gives good separation of very

small amounts, of especially water-soluble small amounts, of especially water-soluble compounds, e.g. sugars.compounds, e.g. sugars.

DISADVANTAGESDISADVANTAGESi.i. Fragile – chromatogram may be destroyed by Fragile – chromatogram may be destroyed by

chemicals used for visualizationchemicals used for visualizationii.ii. May be time-consuming.May be time-consuming.

RF VALUESRF VALUESRf (rate of flow) values are Rf (rate of flow) values are

used as a way of used as a way of identifying compounds identifying compounds on a chromatogram. on a chromatogram.

In theory, the Rf value is In theory, the Rf value is constant in a constant constant in a constant set of chromatographic set of chromatographic conditions. In practice, it conditions. In practice, it is difficult to achieve is difficult to achieve because of the many because of the many factors that may affect factors that may affect the Rf value.the Rf value.

RF VALUES & MOISTURE RF VALUES & MOISTURE

Very NB:= amount of moisture adsorbed on the Very NB:= amount of moisture adsorbed on the thin layer. The presence of significant amounts thin layer. The presence of significant amounts of moisture decrease the number of sites of moisture decrease the number of sites available for active adsorption. The compounds available for active adsorption. The compounds undergoing separation will therefore have a undergoing separation will therefore have a higher than normal Rf value.higher than normal Rf value.

The moisture content of thin layer plates is difficult The moisture content of thin layer plates is difficult to control & is therefore a major factor in non-to control & is therefore a major factor in non-reproducibility of Rf values.reproducibility of Rf values.

RF & TANK SATURATIONRF & TANK SATURATION

In a non-saturated tank, the solvent In a non-saturated tank, the solvent evaporates as it travels up the plate evaporates as it travels up the plate producing a lower solvent front than in a producing a lower solvent front than in a saturated tank.saturated tank.

Rf values of substances being separated will Rf values of substances being separated will therefore be higher than these obtained therefore be higher than these obtained using a saturated tank.using a saturated tank.

Developing the PlatesDeveloping the Plates

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

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

Identifying the SpotsIdentifying the Spots

If the spots can be seen, outline them with a If the spots can be seen, outline them with a pencil.pencil.

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

Identifying the SpotsIdentifying the Spots

Commercial TLC plate after Commercial TLC plate after developmentdevelopmentin normal lightingin normal lighting

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

Interpreting the DataInterpreting the DataThe Rf value for each spot The Rf value for each spot

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

Interpreting the DataInterpreting the Data

(Note: Rf values often depend on the temperature (Note: Rf values often depend on the temperature and the solvent used in the TLC experiment; the and the solvent used in the TLC experiment; the most effective way to identify a compound is to most effective way to identify a compound is to spot known substances next to unknown spot known substances next to unknown substances on the same plate).substances on the same plate).

In addition, the purity of a sample may be In addition, the purity of a sample may be estimated from the chromatogram. An impure estimated from the chromatogram. An impure sample will often develop as two or more spots, sample will often develop as two or more spots, while a pure sample will show only one spot.while a pure sample will show only one spot.

RECAP - PCRECAP - PC

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

PC - DESCRIPTIONPC - DESCRIPTION

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

Preparing the ChamberPreparing the Chamber Choose a developing chamber that can be Choose a developing chamber that can be

sealed well. The chamber should be large sealed well. The chamber should be large enough to hold the paper that is to be enough to hold the paper that is to be developed.developed.

The chamber should be clean and dry before The chamber should be clean and dry before use.use.

Add the mobile phase to the chamber so that Add the mobile phase to the chamber so that it is about 2 cm deep. Seal the chamber it is about 2 cm deep. Seal the chamber tightly and let the chamber stand overnight if tightly and let the chamber stand overnight if possible (Allowing the chamber to stand possible (Allowing the chamber to stand permits its atmosphere to become saturated permits its atmosphere to become saturated with the developing solvent. A saturated with the developing solvent. A saturated atmosphere allows for more effective atmosphere allows for more effective development of the chromatograms. The development of the chromatograms. The larger the chamber, the longer it should larger the chamber, the longer it should standstand ) )

Preparing the ChamberPreparing the Chamber

The larger the The larger the chamber, the longer it chamber, the longer it should stand should stand

Preparing the Stationary PhasePreparing the Stationary Phase

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

Spotting the SamplesSpotting the Samples

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

Spotting the SamplesSpotting the Samples

If a larger quantity of If a larger quantity of sample is needed for the sample is needed for the experiment than is experiment than is provided by one provided by one application, the solution application, the solution may be re-spotted. may be re-spotted.

All spots on the All spots on the chromatogram should be chromatogram should be 2 to 2.5 cm away from the 2 to 2.5 cm away from the edges of the paper and edges of the paper and from each other. from each other.

Developing the ChromatogramsDeveloping the Chromatograms

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

Initially, the chromatogram should be suspended Initially, the chromatogram should be suspended in the chamber without touching the solvent. To in the chamber without touching the solvent. To suspend the chromatogram, to the top of the suspend the chromatogram, to the top of the paper and thread a piece of string throught the paper and thread a piece of string throught the paper clip. Then tape the string to the outside of paper clip. Then tape the string to the outside of the chamber to hold the chromatogram in place. the chamber to hold the chromatogram in place. The paper should hang in the development The paper should hang in the development chamber overnight, if possible.chamber overnight, if possible.

Developing the ChromatogramsDeveloping the Chromatograms

After the After the chromatogram has chromatogram has hung in the chamber, hung in the chamber, immerse the paper's immerse the paper's bottom edge into the bottom edge into the developing solvent.developing solvent.

Allow the Allow the chromatogram to dry chromatogram to dry in a well-ventilated in a well-ventilated area. area.

Identifying the SpotsIdentifying the Spots

If the spots can be seen, outline them with a If the spots can be seen, outline them with a pencil.pencil.

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

KROMATOGRAM MINYAK NILAM DENGAN KOLOM PACKING DAN KOLOM

KAPILER

PERALATAN KROMATOGRAFI GAS

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 interactionsbetween the stationary phase, the moleculesbeing analyzed, and the solvent(s) used.

FASA DIAM : PADATANFASA GERAK : CAIRAN

DIGUNAKAN UNTUK MEMISAHKAN DAN MENGANALISIS CAMPURAN SENYAWA YANG BERSIFAT KURANG VOLATIL

PERALATAN HPLC