ANALISIS HPLC dan METODE DERIVATISASI

INTERAKSI SOLUT-FASE GERAK-FASE DIAM

ADSORPSI PARTISI

PENUKAR ION SIZE EXCLUSION

PASANGAN ION

INTERAKSI SOLUT-FASE GERAK-FASE DIAM

SEC

GFC

LSCRPC

RPCBPC

RPCBPCLSC

IECIPC

LARUT DALAM PELARUT ORGANIK

LARUT DALAM AIR

NON POLAR

POLAR

NON IONIK

IONIK

LARUT DALAM PELARUT ORGANIK

LARUT DALAM AIR

BM>2000

BM<2000

SAMPEL

SKEMA PEMILIHAN SISTEM KROMATOGRAFI

Keterangan :SEC : Size Exclusion ChromatographyGFC : Gel Filtration ChromatographyLSC : Liquid Solid ChromatographyRPC : Reverse Phase ChromatographyBPC : Bonded Phas ChromatographyIEC : Ion Exchange ChromatographyIPC : Ion Pair Chromatography

SKEMA HPLC

Ada banyak faktor yang mempengaruhi efisiensi kolom sehingga harus dioptimasi agar diperoleh pemisahan yang baik, yaitu :•Kecepatan alir fase gerak, kecepatan alir yang sangat lambat akan menyebabkan terjadinya difusi longitudinal, sedangkan bila terlalu cepat akan menyebabkan terjadinya transfer masa non ekuilibrium, sehingga terjadi pelebaran pita kromatogram•Ukuran partikel fase diam, semakin kecil ukuran partikel maka efisiensi semakin baik Tetapi menyebabkan tekanan dalam kolom semakin besra sehingga dibutuhkan kekuatan pompa yang semakin besar.•Panjang kolom, semakin panjang akan semakin besar harga efisiensi kolom, tetapi dapat menyebabkan terjadinya pelebaran pita.•Viskositas fase gerak, semakin kecil harga viskositas fase gerak maka efisiensi kolom semakin besar.•Temperatur, semakin tinggi temperatur maka viskositas semakin rendah dan efisiensi kolom menjadi lebih besar.•Volume ekstra kolom, semakin besar volume ekstra kolom maka kemungkinan terjadinya pelebaran pita semakin besar, sehingga efisiensi semakin berkurang.•Jumlah sampel dan volume sampel, bila jumlah maupun volume sampel sangat besar (overload) maka kemungkinan terjadinya pelebaran pita semakin besar, sehingga efisiensi semakin berkurang.

Agar diperoleh hasil pemisahan yang baik, maka perlu dilakukan evaluasi kolom secara berkala. Evaluasi terhadap kelayakan kolom dapat dipantau dengan mengevaluasi harga beberapa parameter di bawah ini secara berkala :

•Faktor kapasitas berkisar 2 – 10• Jumlah lempeng teoritik tidak mengalami perubahan yang signifikan

•Faktor resolusi (RS) harus > 1.5•Peak asimetri < 1.2•Tekanan kolom dalam kisaran normal (dapat dikerjakan oleh pompa dengan ringan)

VARIABEL PEPTIDA PROTEIN

Kolom

Bonded phase

Dimensi

Partikel

C18 atau C8

0.46 X 15 atau 25 cm

3.5-10 m (diameter)

80-300 A ()

C4, C3, CN

0.46 x 5-15 cm

3.5-10 m

(diameter)

300 A ()

Fase gerak

Solven A

Solven B

Gradien

0.12% TFA/air

0.10%TFA/air

0-60% B/60 menit

0.12% TFA/air

0.10%TFA/air

0-60% B/60 menit

Temperatur 40 – 80 C 40 – 80 C

Kecepatan alir 0.5 – 2 ml/menit 0.5 – 2 ml/menit

Ukuran sampel

Volume

Berat

10-50 L

1-100 g

10-50 L

1-100 g

Contoh Aplikasi Pemisahan Peptida Dan Protein Dengan Rp-HPLC

Problem yang mungkin muncul :•Bentuk pita yang jelek : melebar, tailing•Recovery rendah•Timbul pita yang misterius•Pita ganda untuk satu jenis analit•Performance kolom berubah, tr tidak reprodusibel

Faktor penyebab :•Kolom rusak, terlalu asam, terlalu hidrofob, terlalu kecil ukuran pori-porinya•Denaturasi sampel•Isomerisasi (cis ke trans)

Pengatasannya :•Pemisahan pada pH rendah (fase gerak 0.1 % Tetra fluoro Acid)•Gunakan asetonitril sebagai solven organik. Untuk sampel yang hidrofob gunakan propanol•Analisis dikerjakan pada temperatur kolom 50 - 80c•Gunakan zwitterionic detergent

VARIABEL PROTEIN ASAM

(ANION EXCHANGE)

PROTEIN BASA

(KATION EXCHANGE)

Kolom

Bonded phase

Ukuran

DEAE, SAX,

5-25 x 0.46cm

CM, SP

5-25 x 0.46 cm

Fase gerak

Solven A

Solven B

Gradien

10mm tris atau phosphat (pH 8)

Solvent A + 0.5m NaCl atau Na-

asetat

0-100% B dalam 30 menit

10mm bis-tris atau phosphat (pH

6)

Solvent A + 0.5m NaCl atau Na-

asetat

0-100% B dalam 30 menit

Temperatur 35 c 35 c

Kecepatan alir 1.0ml/menit 1.0ml/menit

Ukuran sampel

Volume

Berat

10-50 l

1-100 g

10-50 l

1-100 g

Contoh aplikasi pemisahan peptida dan protein dengan ion exchange HPLC

Keuntungan :• Konformasi protein tetap terjaga, • Kemungkinan denaturasi kecil, • Dapat digunakan untuk isolasi dan purifikasi protein dengan tetap berbentuk bioaktif

• Protein basa biasa memakai kation exchange, dengan ph 3

Problem yang sering muncul :• Recovery rendah dan dapat diatasi dengan menggunakan gradien elusi dengan fase gerak mengandung garam.

Detector, hendaknya memiliki kriteria : sensitivitasnya tinggi, batas deteksi rendah, linearitas respon tinggi dan reprodusibilitasnya tinggi. Ada banyak detektor yang dapat diaplikasikan, yaitu :• Detektor spektrofotometer UV/Vis, merupakan detektor universal dan dapat diaplikasikan pada semua analit yang dapat menyerap sinar uv/vis.

• Detektor spektrofluorometer, merupakan detektor yang lebih selektif dan lebih sensitif.

• Detektor indeks bias, tergantung pada perubahan harga indeks bias fase gerak oleh karena adanya analit atau solut.

• Detektor elektrokimia, ada banyak jenisnya antara lain : detektor konstanta dielektrika, detektor konduktometer, detektor amperometer.

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DERIVATISASI

ANALIT SENYAWA YANG SENSITIF DITANGKAP DETEKTOR

KOMPLEX

PEMISAHAN LEBIH BAGUSLEBIH SENSITIF

FLUORESCENT

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DERIVATIZATION……

• Derivatization, prior to chromatographic analysis, can be used for a number of reasons in sample preparation, such as.– TO IMPROVE PERFORMANCE OF COMPOUNDS

and ENHANCED DETECTABILITY– TO IMPROVE SENSITIVITY– TO IMPROVE SEPARATION

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DERIVATIZATION…….• Liquid Chromatography:

– fluorescent derivatives can be prepared to render the substances specifically detectable at high sensitivity. dansyl chloride (5-dimethyl aminonaphthalene-1-sulphonyl chloride) is strongly recommended for phenols, and primary and secondary amines. 4-chloro-7-nitrobenz-2,1,3-oxadiazole (NBD chloride) which provides highly fluorescent derivatives of primary and secondary amines but aromatic amines, phenols and thiols only yield weakly or non fluorescent derivatives.

– UV chromaphores are often introduced into sample molecules to increase their sensitivity to UV absorption, for examples: benzoyl chloride, m-toluol chloride and p-nitrobenzoyl chloride are reagents that can add a benzene ring to a solute molecule and render it UV absorbing; 3,5-dinitrophenylhydrazine and p-nitrobenzylhydroxylamine are suitable for a solute containing carbonyl group

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• THERE ARE 3 TYPES OF DERIVATIZATION TECHNIQUE IN HPLC OR CE:– ON-COLUMN

after the HPLC column is equlibrated with mobile phase in the presence of the derivative reagent, the analyte is introduced and then eluated

– PRE COLUMNoften needs complicated procedures and can not handle unstable reaction compounds

– POST COLUMN

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DERIVATIZATION IN HPLC

DERIVATIZATION

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(Carbohydrates analysis by HPLC)….

SugarsPolyhydric alcohols

HPLCREFRACTIVE INDEXION-EXCHANGE

PROBLEM. INSUFFICIENT SENSITIVITY AND SPECIFITY

DERIVATIZATION

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• POST COLUMN:– POST COLUMN WITH P-AMINOBENZOIC ACID HYDRAZIDE

AND DETECTED USING SPECTRO VIS 410 NM– GLUCOSE USING IMMOBILZED (GOD), HYDROGEN

PEROXIDE IS DETECTED WITH ECD• PRE COLUMN– WITH P-NITROBENZOYL CHLORIDE (PNB-Cl), FOLLOWED BY

NORMAL HPLC AND SPECTRO UV 260 NM– WITH PHENYL ISOCYANANTE (PHI), FOLLOWED BY RP HPLC

USING SPECTRO UV 240 NM– WITH ISATOIC ANHYDRIDE TO FORM FLUORESCENT

ANTHRANILOYL, Λex 360 nm λem 420 nm, NORMAL AND RP HPLC

(Carbohydrates analysis by HPLC)….

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• TO IMPROVE UV DETECTION.– BORON IN THE ALKALINE MOBILE PHASE– ADDITION OF SORBIC ACID– DERIVATIZATION WITH 1-PHENYL-3-METHYL-5-

PYRAZOLONE,ETHYL-P-AMINOBENZOATE, 2-AMINOPYRIDINE, FOLLOWED BY UV 240-350 NM

• TO IMPROVE FLUORESCENT DETECTION– 5-AMINONAPHTHALENE-2-SULFONAT (Λex 325 nm; λem

475nm)– 9-AMINOPYRENE-1,4,6-TRISULFONATE λex 475 nm, λem

512nm)

(Carbohydrates analysis by CE)….

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DERIVATIZATION OF AMINO ACID

•WITH HEPTAFLUOROBUTYRIC ANHYDRIDE

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DERIVATIZATION AMINO ACID….. •WITH 6-aminoquinolyl-N-hydroxysccinimidyl carbamate (AQC)

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•WITH O-PHTHALALDEHYDE (OPA)

DERIVATIZATION AMINO ACID…..

OPA REACTS WITH PRIMARY AMINO GROUP IN THE PRESENCE OF THIOL REAGENTOPA DOES NOT REACT SECONDARY AMINO GROUP, SUCH AS PROLINE AND HYDROXYPROLINE

Λex 230 nm, λem 420 nm

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DERIVATIZATION AMINO ACID…..

• POST COLUMN WITH NINHYDRIN

Blue violet

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• WITH NITROBENZOFURAN CHLORIDE (NBD-Cl) FOR SECONDARY AMINO ACID

DERIVATIZATION AMINO ACID…..

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DERIVATIZATION AMINO ACID….. •WITH FMOC-Cl (Fluorenylmethyloxycarbonyl chloride) FOR SECONDARY AA

FMOC IS FLUOROGENIC. THE EXCESS OF FMOC MUST BE REMOVED BY EXTRACTION WITH PENTANEFMOC CAN FORM POLYMER, TO SOLVE THIS PROBLEM ALKALINE TREATMENT IS CARRIED OUT

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• FATTY ACIDS ARE AN IMPORTANT ENERGY SOURCE• ESSENTIAL FATTY ACIDS: LINOLEIC ACID AND

ARACHIDONIC ACID• FATTY ACIDS OFTEN NEED TO BE QUANTIFIED AS

INDICATORS OF RANCIDITY, FRESHNES AND ADULTERATIN

• DETERMINATION:– GC AND HPLC AFTER DERIVATIZATION (WITH

METHYLENE BLUE)– GC AFTER METHYL ESTERIFICATION

DERIVATIZATION FATTY ACID

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• DERIVATIZATION WITH METHYLENE BLUE: FATTY ACIDS ARE EXTRACTED AS ION-PAIRS WITH CHLOROFORM FROM AQUOEOUS ACETONITRILE MOBILE PHASE AFTER THE POST COLUMN ADDITION OF AQUEOUS METHYLENE BLUE SOLUTION

• DERIVATIZATION WITH 2-NITROPHENYLHYDRAZINE HCl (2-NPH) AND FOLLOWED BY RP-HPLC AND DTECTED AT 400NM.

DERIVATIZATION FATTY ACID……

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• VITAMIN B1 (THIAMINE)– POST AND PRE COLUMN RP-HPLC WITH THIOCHROME FLUORESCENE (λex 375

nm, λex 440 nm)

• VITAMIN B6 (PYRIDOXINE)– POST COLUMN WITH SULFUROUS ACID AFTER EXTRACTION WITH PERCHLORIC

ACID AND SEPARATION WITH ION-PAIRED RP-HPLC

• VITAMIN C (ASCORBIC ACID)– THE REDUCED AND OXIDIZED FORM OF VITAMIN C ARE REFFERED TO AS

ASCORBIC ACID (AA) AND DEHYDROASCORBIC ACID (DHAA). All of them are active

– OXIDIZATION OF AA TO DHAA FOLLOWED BY DERVATIZATION WITH DFQ 3-(1,2-DIHYDROXYETHYL)FLUORO[3,4-b]QUINOXALINE-1-ONE, AND THEN SEPARATION ON RP-HPLC AT λex 350 nm AND λem 430 nm

DERIVATIZATION VITAMINS

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ANALYSIS OF VITAMIN C

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FOLACIN (PTEROYLGLUTAMIC ACID)

DERIVATIZATION VITAMINS…..

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• BIOTIN (VITAMIN B7)– PRE COLUMN HPLC USING 4-BROMOMETHYL-7-

METHOXYCOUMARIN (Br-MMC)– POST COLUMN WITH CHLORINATION OF THE AMIDE

GROUP OF BIOTIN; OR INTRODUCTION OF THIAMINE TO FORM THIOCHROME AND DETECTION WITH FLUORESCENT.

DERIVATIZATION VITAMINS……

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• VITAMIN K– Naturally occuring vitamin K is vit K1

(phytonadione or phylloquinones) found in plants and vit K2 (menaquinones) found in bacteria and vitamin K3 (menadiones)

– POST COLUMN REDUCTION OF VITAMIN K WITH ZINC OR PLATINA TO FORM FLUORESCENCE PRODUCT (CORRESPONDING HYDROQUINONES) AND FLUORESCENT DETECTION

DERIVATIZATION VITAMINS……

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DERIVATIZATION VITAMINS……

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• SACCHARIN, GLYCYRRHIZIC ACID, ASPARTAME AND SORBITOL

• ASPARTAME:

DERIVATIZATION OF SWEETENERS

ASPARTAME CAN BE ANALYZED USING PRE COLUMN METHOD WITH FLUORESCAMINE TO FORM FLUORESCENT DERIVATIVES FOLLOWED BY RP-HPLC AND DETECTED AT λex 390 nm AND λem 480 nm

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ACESULFAME K

CYCLAMATE, SACCHARIN AND ACESULFAME K CAN BE DETECTED USING POST-COLUMN ION PAIR EXTRACTION AS DESCRIBED IN ANALYSIS FATTY ACID

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DERIVATIZATION OF EMULSIFIERS

• MONOGLYCERIDE (MGL), SUCROSE ESTER OF FATTY ACID (SuE), PROPYLENE GLYCOL ESTER OF FATTY ACID (PGE), SORBITAN ESTER OF FATTY ACID

• ANALYSIS OF MGL, SuE, PGE USES 3,5-DINITROBENZOIL CHLORIDE WITH PRE COLUMN METHOD TO FORM DNBC DERIVATIVES FOLLOWED BY SEPARATION WITH RP-HPLC AND UV DETECTION AT λmax 230 nm

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SUCROSE ESTER OF FATTY ACID