KROMATOGRAFI

Pertemuan ke 9

KROMATOGRAFI• Kromatografi adalah suatu cara pemisahan senyawa dari campurannya melalui elusi

sepanjang fasa lain yang tak bergerak.

• Eluen dapat berupa fasa gas atau cair yang selanjutnya disebut fasa gerak (mobile phase), sedangkan fasa tak bergerak atau fasa stasioner (fasionery phase) dapat berupa padatan atau cairan yang diselaputkan pada permukaan padatan pendukung (supporting solid) yang innert.

• Chromatography involves a sample (or sample extract) being dissolved in a mobile phase (which may be a gas, a liquid or a supercritical fluid).

• The mobile phase is then forced through an immobile, immiscible stationary phase. The phases are chosen such that components of the sample have differing solubilities in each phase.

• A component which is quite soluble in the stationary phase will take longer to travel through it than a component which is not very soluble in the stationary phase but very soluble in the mobile phase. As a result of these differences in mobilities, sample components will become separated from each other as they travel through the stationary phase.

SEJARAH KROMATOGRAFI

• 1834 Runge F F :pemisahan campuran zat warna dan ekstrak tanaman

• 1906 Tswett M. : pemisahan pigmen kloropil pada CaCO3 dengan eluen

petroleum eter → kromatografi

• 1931 Kuhn, R et al. : kromatografi cair-padat, pemisahan xanthophyl kuning telur

• 1941 Tiselius A.: kromatografi cair, frontal analisis

• 1941 Martin & Synge : model efisiensi kolom

• 1944 Consden, Gordon, Martin : kromatografi kertas

• 1947 Boyd et al.: kromatografi penukar ion

• 1951 Kirchner : Kromatografi lapis tipis (TLC)

• 1952 James & Martin : Kromatografi gas

• 1956 van Dempter : fungsi distribusi gausian

• 1964 J C. Moore : kromatografi gel permeation

Klasifikasi kromatografi

Fasa gerak Fasa diam Mekanisme Istilah/nama

Gas padat adsorbsi GSC

Cair partisi GLC

Cair cair partisi LLC

padat adsorbsi LSC

Penukar ion Penukar ion IEC

Gel Exclusion EC

Cairan superkritik

Spesi organik pada padatan

partisi SFC

Kromatografi

Kromatografigas

Kromatografi cair

Gas - Cair Gas - padat Liqiud-liquid Lquid-solid Ion-exchange Exclusion

Klasifikasi : peralatan

• Kromatografi kolom

• Kromatografi lapis tipis (TLC = Thin Layer Chromatography)

• Kromatografi kertas (paper chromatography)

• Kromatografi gas (GC= gas chromatography)

• Kromatografi cair kinerja tinggi (HPLC = high performance liquid chromatography)

KROMATOGRAFI KOLOM

Kromatografi Kolom

Kesetimbangan distribusi

Fasa gerak

Fasa diam

K = -----------

Cd

CgK = koefisien distribusiCg = Konsentrasi dalam fasa gerakCd = konsentrasi zat dalam fasa diam

Fraksi waktu tinggal dalam fasa gerak =------------------------------------------------- Jumlah molekul dalam fasa gerak

Total molekul yang ada

= ------------------------Cg Vg

Cg Vg + Cd Vd

= ------------------------ 1

1 + K Vd/Vg

= ----------- 1

1 + k’ k’ = faktor kapasitas

Vg =volume fasa gerak

Laju pergerakan

Fraksi = ----------- 1

1 + k’

Laju linier =

Laju pergerakan = ----------- 1

1 + k’

Laju pergerakan rata-rata ,molekul ditentukan oleh:

1.Laju fasa gerak2.Rasio volume fasa diam terhadap volume fasa gerak3.Koefisien distribusi: khas untuk setiap komponen

Waktu retensi, tR

Waktu yang diperlukan oleh komponen menempuh perjalanan sepanjang kolom

tR = ------------------ = -------- (1+k’) panjang lintasan

Laju pergerakan

L

= tM (1+k’)

tM = waktu yang diperlukan oleh molekul fasa gerak sepanjang lintasan/kolom

Volume retensi

Volume fasa gerak yang diperlukan untuk mengelusi komponen sampel

Vol = waktu x laju alir

VR = tR x F

VR = VM (1+k’) = VM + KVd

Retensi relatif:

Kromatografi lapis tipis TLC

Thin-layer chromatography (TLC) is a chromatographic technique that is useful for separating organic compounds. Because of the simplicity and rapidity of TLC, it is often used to monitor the progress of organic reactions and to check the purity of products.

Teknik Elusi KLT/KK

Elusi Ascending : dari bawah ke atasElusi Discending : dari atas ke bawah

Elusi dua dimensi

Gambar teknik elusi

Elusi dua dimensi

Two-dimensional TLC uses the TLC method twice to separate spots that are unresolved by only one solvent. After running a sample in one solvent, the TLC plate is removed, dried, rotated 90o, and run in another solvent. Any of the spots from the first run that contain mixtures can now be separated. The finished chromatogram is a two-dimensional array of spots.

Identifikasi spot hasil elusi

• Spot berwarna• Spot tak berwarna

Spot tak berwarna:

- Diuapi dengan amoniak

- Diuapi dengan iodium (I2)

- Disemprot dengan larutan pengembang (pewarna)

- Disemprot dengan asam sulfat pekat

Nilai Rf

Aplikasi pemisahan TLC

No Senyawa yang dipisahkan

Solven (Fasa gerak) Sorben (fasa diam) Reagent deteksi

1. Kloroplast pigmens

Isooktan-aceton-eter (3:1:1) -Silika gel-Polyamida

-

Petroleum eter-aseton (4:6) Alumina

2. Alkaloid Bensen-etanol (9:1) Silika gel Bromocresol greenDragendorffIodineCHCl3-aseton-dietilamin (5:4:1) Silika gel

3. Amina Etanol 90% - NH3 25% (4:1) Silika gel AsetoasetilfenolAlizarin

Aseton-H2O (99:1) Kielsguhr G

4. Gula Benzena-as.asetat-metanol (1:1:3)

Silika gel dg bufer as. borat

Butanol-piridin-H2O (6:4:3) Selulosa

5. Food Dyes Metil etil keton-as asetat-metanol (40:5:5)

Silika gel G

Butanol-etanol-H2O (9:1:1) Alumina

No Senyawa yang dipisahkan Solven (Fasa gerak) Sorben (fasa diam)

6. Tserpenoid Heksana-etil asetat (85:15) -pati-asam asalisilat

Benzena petroleum eter Alumina

7. Vitamin Metanol, CCl4, atau petroleum eter Alumina

Metanol, propanol atau CHCl3 Silika gel G

8. Fenol Benzena, atau dietil eter alumina

Heksana-etilasetat (4:1 atau 3:2) Silkika gel + as oksalat

9. Asam amino Butanol-asamasetat-H2O (4:1:1) Silika gel G

N-butanol-aseton-NH3-H2O (10:10:5:2) Selulosa

Iso propanol-asam format-H2O (20:1:5) selulosa

10. Flavonoid dan coumarin

Metanol-H2O (8:2 atau 6:4) Polyamida

Petroleum eter – etil asetat (2:1) Silika gel G

11. Steroid dan sterol Benzene-etil asetat (9:1) Silika gel G

CHCl3 – etanol (96:4) alumina

Zweig G & Joseph S, 1972, Handbook of Chromatography, Vol II. CRC Press USA

Pereaksi pengembang (detection Reagent)

Aplikasi pemisahan

Kromatografi Kertas

Aplikasi Pemisahan

Kromatografi Gas

Gas Chomatography

GC

PRINSIP GAS-KROMATOGRAFI

• Gas chromatography - specifically gas-liquid chromatography - involves a sample being vapourised and injected onto the head of the chromatographic column. The sample is transported through the column by the flow of inert, gaseous mobile phase. The column itself contains a liquid stationary phase which is adsorbed onto the surface of an inert solid.

Ekstraksi

Skema peralatan GC

Plotter → kromatogram

1

2

3

45

Carrier gas

- The carrier gas must be chemically inert.

- Commonly used gases include nitrogen, helium, argon, and carbon dioxide.

- The choice of carrier gas is often dependant upon the type of detector which is used.

- The carrier gas system also contains a molecular sieve to remove water and other impurities.

Sample injection port

For optimum column efficiency, the sample should not be too large, and should be introduced onto the column as a "plug" of vapour - slow injection of large samples causes band broadening and loss of resolution.

Microsyringe is used to inject sample through a rubber septum into a flash vapouriser port at the head of the column.

The temperature of the sample port is usually about 50°C higher than the boiling point of the least volatile component of the sample.

For packed columns, sample size ranges from tenths of a microliter up to 20 microliters. Capillary columns, on the other hand, need much less sample, typically around 10-3 mL. For capillary GC, split/splitless injection is used. Have a look at this diagram of a split/splitless injector;

A small amount of liquid (microliters) is injected through a silicon rubber septum (using a special microliter syringe) into the hot (usually 200+ degrees C) GC injector that is lined with an inert glass tube. The injector is kept hot by a relatively large, metal heater block that is thermostatically controlled.

The injector can be used in one of two modes; split or splitless. The injector contains a heated chamber containing a glass liner into which the sample is injected through the septum. The carrier gas enters the chamber and can leave by three routes (when the injector is in split mode). The sample vapourises to form a mixture of carrier gas, vapourised solvent and vapourised solutes. A proportion of this mixture passes onto the column, but most exits through the split outlet. The septum purge outlet prevents septum bleed components from entering the column.

InjectorDiagram skematik

Columns• There are two general types of column, packed and capillary (also known as open tubular). • Packed columns contain a finely divided, inert, solid support material (commonly based on

diatomaceous earth) coated with liquid stationary phase. Most packed columns are 1.5 - 10m in length and have an internal diameter of 2 - 4mm.

• Capillary columns have an internal diameter of a few tenths of a millimeter. They can be one of two types; wall-coated open tubular (WCOT) or support-coated open tubular (SCOT). Wall-coated columns consist of a capillary tube whose walls are coated with liquid stationary phase. In support-coated columns, the inner wall of the capillary is lined with a thin layer of support material such as diatomaceous earth, onto which the stationary phase has been adsorbed. SCOT columns are generally less efficient than WCOT columns. Both types of capillary column are more efficient than packed columns.

Kolom Kapiler dalam oven

Kolom packing Kolom kapiler

Column temperature

For precise work, column temperature must be controlled to within tenths of a degree.

The optimum column temperature is dependant upon the boiling point of the sample. As a rule of thumb, a temperature slightly above the average boiling point of the sample results in an elution time of 2 - 30 minutes. Minimal temperatures give good resolution, but increase elution times. If a sample has a wide boiling range, then temperature programming can be useful. The column temperature is increased (either continuously or in steps) as separation proceeds.

capillary gas chromatography : high resolution. Capillary columns : 1) more theoretical plates per meter as compared to packed columns and 2) since they have less resistance to flow they can be longer than packed

columns. This means that the average capillary column (30 meters long) has approximately 100,000 theoretical plates while the average packed column (3 meters) has only 2500 plates

But with this separation power comes some limitations: 1) Capillary columns, because they have smaller diameters (0.05 to 0.53 mm) than packed columns (2 to 4 mm), require relatively specialized injectors and ancillary flow and pressure controllers and 2) capillary column require a smaller amount of sample than packed columns. While the average sample mass of each component in a mixture that is separable by packed column GC can be in the microgram range (10-6 grams) per injection, capillary columns routinely only handle 50 nanograms (10-9 grams) of a particular component or less.

DETEKTOR

FID = Flame ionisation detector

ECD = Electron chapture

FPD = Flame photometry detector

AED = Atomic Emission Detector

PID = Photo ionisation detector …

There are many detectors which can be used in gas chromatography. Different detectors will give different types of selectivity. A non-selective detector responds to all compounds except the carrier gas, a selective detector responds to a range of compounds with a common physical or chemical property and a specific detector responds to a single chemical compound.

Detectors can also be grouped into concentration dependant detectors and mass flow dependant detectors. The signal from a concentration dependant detector is related to the concentration of solute in the detector, and does not usually destroy the sample Dilution of with make-up gas will lower the detectors response. Mass flow dependant detectors usually destroy the sample, and the signal is related to the rate at which solute molecules enter the detector. The response of a mass flow dependant detector is unaffected by make-up gas. Have a look at this tabular summary of common GC detectors:

Detectors

Detector

Type Support gases

Selectivity Detectability Dynamic range

FID Mass flow Hydrogen and air

Most organic cpds. 100 pg 107

TCD Concentration Reference Universal 1 ng 107

ECP Concentration Make-up Halides, nitrates, nitriles, peroxides, anhydrides, organometallics

50 fg 105

FPD Mass flow Hydrogen and air possibly oxygen

Sulphur, phosphorus, tin, boron, arsenic, germanium, selenium, chromium

100 pg 103

PID Concentration Make-up Aliphatics, aromatics, ketones, esters, aldehydes, amines, heterocyclics, organosulphurs, some organometallics

2 pg 107

NPD Mass flow Hydrogen and air

Nitrogen, phosphor 10 pg

FID

ECD

FPD

Photodiode dan photovoltaic diode

PID

AED

Kondisi Operasional analisis

Jenis kolom

Bahan kolom (fasa diam)

Laju alir gas pembawa (carrier gas)

Suhu injector

Suhu kolom

Mode pemanasan kolom (isotermal atau temperatur terprogram)

Jenis detektor

Suhu detektor

Hasil Kromatografi Gas

• Berupa kromatogram• Aspek kualitatif (nilai Rt)• Aspek kuantitatif

Metoda Standar:

Metoda standar eksternal Internal addisi

Standar eksternal

• Sampel dibuat kromatogram diinjeksikan sendiri)

• Standar dibuat kromatogram• Dibandingkan antara nilai Rt standar dan Rt

sampel. Peak atau puncak sampel yang memiliki nilai Rt yang sama berarti senyawa sampel sama dengan senyawa standar

Sandar internal

• Sampel dibuat kromatogram diinjeksikan sendiri)

• Standar + sampel dicampur dan dibuat kromatogram

• Puncak/peak sampel dan peak campuran diperbandingkan. Peak yang semakin tinggi menunjukkan senyawa sampel + sandar.

Liquid Chromatography (LC) Liquid chromatography (LC) is an analytical

chromatographic technique that is useful for separating ions or molecules that are dissolved in a solvent. If the sample solution is in contact with a second solid or liquid phase, the different solutes will interact with the other phase to differing degrees due to differences in adsorption, ion-exchange, partitioning, or size. These differences allow the mixture components to be separated from each other by using these differences to determine the transit time of the solutes through a column.

HPLC

Kolom HPLC

Waktu Retensi

Retention factor, k', is often used to describe the migration rate of an analyte on a column. You may also find it called the capacity factor. The retention factor for analyte A is defined as;

k'A = t R - tM / tM

t R and tM are easily obtained from a chromatogram.

The time taken for the mobile phase to pass through the column is called tM.

TEORI PLAT

HETP = L / N

Kromatogram

Resolusi dan Selektivitas

selectivity factor, a , which describes the separation of two species (A and B) on the column;

a = k 'B / k 'A

Jumlah plat

TEORI LAJU ALIR

HETP = A + B / u + C u

where u is the average velocity of the mobile phase. A, B, and C are factors which contribute to band broadening.

A - Eddy diffusionThe mobile phase moves through the column which is packed with stationary phase. Solute molecules will take different paths through the stationary phase at random. This will cause broadening of the solute band, because different paths are of different lengths.

B - Longitudinal diffusionThe concentration of analyte is less at the edges of the band than at the center. Analyte diffuses out from the center to the edges. This causes band broadening. If the velocity of the mobile phase is high then the analyte spends less time on the column, which decreases the effects of longitudinal diffusion.

C - Resistance to mass transferThe analyte takes a certain amount of time to equilibrate between the stationary and mobile phase. If the velocity of the mobile phase is high, and the analyte has a strong affinity for the stationary phase, then the analyte in the mobile phase will move ahead of the analyte in the stationary phase. The band of analyte is broadened. The higher the velocity of mobile phase, the worse the broadening becomes.

Metoda Analisis

• Kondisi operasional alat• Aspek kuantitaif: standar

- Standar ekxternal- Standar internal- Standar adisi

Hasil Analisis Kromatografi

Aspek Kualitatif

Aspek kuantitatif

Aplikasi Analisis kromatografi