Aplikasi penggunaan hplc

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1 Aplikasi penggunaan hplc
Yunika mayangsari, M. Biotech. Fakultas Teknologi Pertanian, UGM

2 Aplikasi hplc pada analisis
gula Asam amino Asam organik Metabolit sekunder: aflatoksin (b1, g1, b2, g2) dan okratoksin a Fakultas Teknologi Pertanian, UGM

3 APLIKASI HPLC UNTUK ANALISIS GULA
Gula dikenal sebagai komponen organik yang dikenal melimpah di alam. Berbagai jenis/macam gula tersedia di sekitar kita seperti monosakarida, oligosakarida, polisakarida gula alami, gula yang diasamkan,gula amina, gula alkohol, dan berbagai macam isomer gula lainnya. Sistem HPLC biasanya digunakan untuk memisahkan dan menganalisis gula. Pemilihan metode pemisahan dan deteksi yang tepat diperlukan agar memperoleh hasil sesuai yang diharapkan. Fakultas Teknologi Pertanian, UGM

4 Beberapa mode/cara/metode separasi/pemisahan yang diaplikasikan pada komponen Gula
■ Size Exclusion: ukuran molekul ■ Ligand Conversion: kemampuan untuk membentuk kompleks dengan ion logam. ■ Partition : kemampuan untuk terpisah di dalam fase stasioner (atau fase geraknya) ■ Anion Exchange: kemampuan untuk bertukar ion ■ Borate Complex Anion Exchange: kemampuan untuk membentuk kompleks dengan borat (pertukaran ion) Borates are the name for a large number of boron-containing oxyanions Fakultas Teknologi Pertanian, UGM

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6 Size Exclusion Metode ini digunakan untuk memisahkan gula berdasarkan pada berat molekulnya. Mode ini dapat digunakan untuk distribusi berat molekul dari ratusan hingga jutaan Dalton. Dikarenakan pemisahan pd mode ini berdasarkan ukuran molekul maka komponen dengan berat molekul yang sama tidak dapat dipisahkan. Polimer hidrofilik digunakan sebagai material pengisi kolom., dan hanya air saja yang digunakan sebagai fase geraknya. Walaupun demikian, garam seringkali ditambahkan pada fase gerak sebagai komponen ionik untuk memebantu interaksi dengan material pengisi (fasa diam). Contoh kolom: aminex Fakultas Teknologi Pertanian, UGM

7 Partition Metode ini cocok untuk memisahkan gula dari monosakarida hingga oligosakarida dan dapat digunakan untuk memisahkan dua tipe gula sekaligus. Material pengisi yang biasanya digunakan adalah aminopropil yang terikat pada polimer silika. Fasa gerak yang sering digunakan adalah asetonitril dan air. Pada metode ini, waktu retensi dari gula disebabkan oleh pemisahan gula dari air yang disebut sebagai proses “kondensasi” di dalam fase diam. Akan tetapi hal yang perlu diperhatikan disini adalah radikal aldehid di dalam gula dapat bereaksi dengan radikal amino dari fasa diam membentuk basa, yang terkadang dapat menyebabkan tailing pada peak untuk pentasakarida (seperti arabinose dan ribose). Hal ini dapat dihambat/dicegah dengan cara menambahkan gula pada fasa gerak. Fakultas Teknologi Pertanian, UGM

8 Ligand Conversion Metode ini menggunakan gel sulfonat polistriren dengan logam penukar ion, seperti Sodium (Na), Calcium (Ca) atau Timbal (Pb) sebagai material pengisi (fasa diam) dan kolom jenis ini dapat digunakan utntuk memisahkan gula hingga disakarida. Waktu retensi molekul gula berdasarkan metode ini didasarkan pada gugus/grup hidroksil dari gu;a dan penukar ion logam. Fasa gerak : air saja (atau mungkin ditambah garam) In addition, it is difficult to mutually separate disaccharides using this method. With Ca or Pb type, care must be used in setting the column temperature to 80 °C (to prevent peaks from splitting or deforming due to anomer separation). Fakultas Teknologi Pertanian, UGM

9 Anion Exchange Dikarenakan nilai pKa gula netral adalah sekitar 12, gula netral dapat ditahan didalam polimer penukar ion apabila menggunakan fasa gerak berupa basa kuat. Pada beberapa kasus, sekitar 0.1 M larutan sodium hidroksida digunakan sebagai fasa gerak. Pada umumnya, metode ini dapat diaplikasikan pada monosakarida hingga oligosakarida. Gula dan komponen polioksi lainnya dapat dengan cepat bereaksi dengan garam borat untuk membentuk kompleks ion negative. Dengan kata lain, gula dapat dipisahkan dengan penukar ion apabila menggunakan larutan buffer borat sebagai fasa geraknya. Fakultas Teknologi Pertanian, UGM

10 Analytical Methods for Amino Acids
HPLC is the most popular method for analyzing amino acid components, which have currently gained attention due to the boom in health foods. This page discusses mainly the detection methods used to analyze amino acids. Fakultas Teknologi Pertanian, UGM

11 Types of Detection Methods
Using UV detection for amino acids in most cases requires using the absorption of the carboxyl group (-COOH) in the 200 to 210 nm range. Some amino acids with benzene rings can also be detected in the 250 to 280 nm range, but in general, they are difficult to analyze as-is with sufficient sensitivity and selectivity. Consequently, derivatization methods have long since been used. Since many amino acids contain amino groups (-NH2 and -NHR) in their structures, a derivatizing reagent that selectively reacts with the amino group is used. Fakultas Teknologi Pertanian, UGM

12 Pre-column Derivatization Method (pre-label method)
n pre-column derivatization, the amino acids are derivertized before injection, and then the reaction products are separated and detected. This concept is illustrated on the right. The "labels" shown indicate the derivatizing reagent. The advantages of this method are as follows: Allows increasing sensitivity by using more expensive reagents that provide lower background levels (than post-column derivatization). Even if unreacted derivatizing reagent is detected, as long as it is separated in the column, it does not cause a problem. Fakultas Teknologi Pertanian, UGM

13 Pre-column Derivatization
On the other hand, a disadvantage is that because the derivatizing reagent is mixed directly with the sample, reaction efficiency (yield) is easily influenced by sample matrix (such as coexisting components and solvent types). Therefore, pre-column derivatization can be considered appropriate when intending to analyze a somewhat limited variety of samples with high sensitivity. Pre-column derivatizing reagents most typically used for amino acid analysis are o-phthalaldehyde, phenyl isothiocyanate (PITC), fluorescamine, and dansyl chloride. he following is an example using a Prominence HPLC system and a Shim-pack XR- ODS reversed-phase column to separate PTC amino acid derivatives (derivatized using a PITC reagent). Fakultas Teknologi Pertanian, UGM

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16 Post-column Derivatization Method (post-column reaction detection method)
The post-column derivatization method involves separating the amino acids in the column, then delivering and mixing the derivatizing reagent to let it react with the amino acids, before finally sending the products to the detector. A flow line diagram of a typical post-column derivatization process is shown to the right. The advantages of this method are as follows: Can be automated offering excellent quantitative performance and reproducibility.  Since sample components are separated before the reaction, enabling it to be used for a wide range of samples. Fakultas Teknologi Pertanian, UGM

17 Post-column Derivatization
Currently, there are mainly two types of reagents available for use in analyzing amino acids, ninhydrin and o-phthalaldehyde. The former is for visible absorption detection and the latter is for fluorescence detection. Fakultas Teknologi Pertanian, UGM

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19 Amino acids (cont) For highly-hydrophilic amino acids, reversed-phase chromatography might not provide adequate retention or selectivity. Using the cation exchange method efficiently separates multiple amino acids. In this way, combining cation exchange chromatography, which is able to separate amino acids from each other or from other amines, together with post- column derivatized detection, which is able to selectively react with amino acids, makes an ideal "marriage" of methods. Fakultas Teknologi Pertanian, UGM

20 Analytical Methods for Organic Acids
Separation Methods The three main separation modes for organic acids are anion exchange, ion exclusion, and reversed-phase. Fakultas Teknologi Pertanian, UGM

21 ion Exchange Mode This separation mode can be thought of as the negative ions in the mobile phase and the organic acid ions (negative ions) competing for the positive ions in the packing material. Many organic acids require using gradient elution for separation, which also increases the analysis time. Fakultas Teknologi Pertanian, UGM

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23 Ion Exclusion Mode This is the most commonly used mode for organic acid analysis. organic acids, the degree they can penetrate the pores is determined by the size of the charge (think of it as pKa), which results in differences in elution time (separation). In the example shown below to the right, acids with large negative charges (small pKa), such as citric and lactic acids, are subject to a larger electrostatic exclusion effect than acids such as acetic acid and, therefore, are eluted more quickly. Consequently, in principle, organic acids elute in order of pKa size, starting with the lowest pKa values, and finish eluting by the time neutral substances start eluting (position for elements that fully penetrated the pores). However, in actuality, a hydrophobic interaction also occurs with some of the packing substrate (styrene- divinylbenzene), which can delay the elution of hydrophobic organic acids in particular. For example, valeric acid elutes much later than acetic acid even though there is little difference in their pKa values. This is because valeric acid is retained more strongly due to a hydrophobic interaction with the substrate material. In this way, a combination of three factors - electrostatic exclusion due to negatively-charged stationary phase, hydrophobic interaction with the stationary phase matrix, and penetration into stationary phase pores, determine elution times. This mode is widely used because it is a simple method that requires minimal adjustment of mobile phase parameters.

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25 Reversed-Phase Mode This mode offers the widest applicability for HPLC, but it is not very widely used for analyzing organic acids. That is because organic acids are hydrophilic, which can often prevent adequate retention or selectivity. However, commercial reversed-phase columns that can sufficiently retain even highly- hydrophilic substances have recently become available on the market. Fakultas Teknologi Pertanian, UGM

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27 Analisis metabolit sekunder dengan metode kromatografi cair kinerja tinggi (HPLC)
Mycotoxin:  Aflatoksin b1, g1, b2 dan g2 Okratoksin Merupakan metode tidak baku yang telah divalidasi oleh tim lab uji mikotoksin, kbphp, tphp, ftp, ugm. (Intruksi kerja uji) Fakultas Teknologi Pertanian, UGM

28 Terima kasih Fakultas Teknologi Pertanian, UGM