LIPID ANALYSIS PENDAHULUAN ANALISIS HASIL PERTANIAN METODE ANALISIS. Lipid Definition: Lipid Classification: Importance of Lipid Analysis:

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ANALISIS HASIL PERTANIAN

Jurusan Teknologi Hasil Pertanian Fakultas Pertanian Universitas Lampung

LIPID ANALYSIS

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P E N D A H U L U A N

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Lipid Definition:

• Komponen organik yang tidak larut air?,

• Mono dan digliserida - Larut pelarut polar dan non polar, • C2-C4 - Larut dalam air.

• US FDA - komponen organik yang tersusun asam lemak C2-C24, ditung sebagai trigliserida.

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Lipid Classification:

• Lipid sederhana (Simple lipid), • Lipid komplek (Compound lipid), • Lipid Turunan (Derivate lipid).

Analisis Lipid . . .

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Analisis Lipid: Pendahuluan . . .

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Importance of Lipid Analysis:

• Akurasi pelabelan kemasan. • Pemenuhan standar identitas.

• Memastikan produk memenuhi standar perusahaan. Catatan: Analisis kadar lemak/minyak yang tidak tepat menghasilkan produk yang sesuai dengan standar dan menyebabkan kerugian yang besar

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General Considerations:

• Minyak/lemak tidak larut dalam air, pemilihan pelarut sangat penting.

• Glycolipid larut dalam alkohol (polar), trigliserida larut dalam heksan dan P. eter (non polar). Pemilihan pelarut disesuaikan dengan jenis lipid dominan.

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M E T O D E A N A L I S I S

A. Metode Ekstraksi Pelarut:

1. Metode Ekstraksi Kontinyu (Goldfish Method). 2. Metode Ekstraksi Semi Kontinyu (Soxhlet Method). 3. Metode Ekstraksi Diskontinyu (Mononnier Method)

D. Metode Ekstraksi Tanpa Pelarut:

1. Babcock Method. 2. Gerber Method.

C. Metode Instrumen:

1. Gas Chromatography Method. 2. Infra Red Method.

3. Specific Gravity Method.

4. Nuclear Magnetic Resonance (NMR) Method.

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Analisis Lipid: Metode . . .

A. METODE EKSTRAKSI PELARUT

• Ekstraksi dengan pelarut organik atau dengan hidrolisis

asam/basa.

• Bergantung pada persiapan sampel dan jenis pelarut yang digunakan.

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Persiapan Sampel:

• Mempengaruhi validitas pengujian. • Disesuaikan dengan jenis sampel.

• Tahapan persiapan sampel:

a. Pengeringan. b. Pengecilan ukuran. c. Hidrolisis

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Analisis Lipid : Ekstraksi pelarut . . .

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Pemilihan Pelarut:

• Harus mempunyai “solvent power” yang tinggi terhadap lipid.

• Titik didih rendah, tidak meninggalkan residu, tidak mudah terbakar, dan tidak beracun.

• Etil eter, petrolium eter, heksana, dan pentana. • Kombinasi 2 atau 3 jenis pelarut digunakan untuk

ekstraksi.

• Pelarut dalam kondisi mur i dan bebas peroksida. • Rasio pelarut dan bahan terlarut penting,

memaksimalkan ekstraksi.

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Jurusan Teknologi Hasil Pertanian Fakultas Pertanian Universitas Lampung 7

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Pemilihan Pelarut:

• Harus mempunyai “solvent power” yang tinggi terhadap lipid.

• Titik didih rendah, tidak meninggalkan residu, tidak mudah terbakar, dan tidak beracun.

• Etil eter, petrolium eter, heksana, dan pentana. • Kombinasi 2 atau 3 jenis pelarut digunakan untuk

ekstraksi.

• Pelarut dalam kondisi mur i dan bebas peroksida. • Rasio pelarut dan bahan terlarut penting,

memaksimalkan ekstraksi.

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Jurusan Teknologi Hasil Pertanian Fakultas Pertanian Universitas Lampung 8

• Sampel diletakan dalam timbel keramik.

• Pelarut secara kontinyu mengekstrak sampel di tibel keramik yang ditempatkan dalam gelas beaker (beaker extraction).

• Setelah waktu ekstraksi selesai (4 jam), timbel dikeluarkan dari beaker, beaker dikeringkan hingga konstan.

• Kadar lemak ditentukan dengan menghitung perubahan berat sampel.

• Keuntungan: cepat dan lebih efisien.

• Kerugian: dapat menyebabkan “channeling”, mengebabkan esktraksi tidak sempurna.

1. Metode Ekstraksi Kontinyu (Goldfish Method)

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Goldfish Fat Extractor

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Analisis Minyak/Lemak: Metode Analisis . . .

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• Pelarut terkumpul pada ruang ekstraksi selama 5-10 menit, merendam sampel, kemudian masuk kembali ke dalam labu didih dengan membawa ekstrak.

• Memerlukan waktu lebih lama dibandingkan dengan sistem kontinyu.

• Digunakan untuk analisis:

a. Lemak sereal (AOAC No. 920.39C). b. Lemak daging (AOAC No. 960.39).

2. Metode Ekstraksi Semi Kontinyu (Soxhlet

Method)

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Soxhlet Fat Extractor

Chapter 8 • Fat Analysis 123

8.3.4 Semicontinuous Solvent Extraction Method: Soxhlet Method

The Soxhlet method (AOAC Method 920.39C for

Cereal Fat; AOAC Method 960.39 for Meat Fat) (8) is

an example of the semicontinuous extraction method and is described below.

8.3.4.1 Principle and Characteristics

For semicontinuous solvent extraction, the solvent builds up in the extraction chamber for 5–10 min and completely surrounds the sample and then siphons back to the boiling flask. Fat content is measured by weight loss of the sample or by weight of the fat removed.

This method provides a soaking effect of the sample and does not cause channeling. However, this method requires more time than the continuous method. Instrumentation for a more rapid and auto-mated version of the Soxhlet method is available (e.g.,

SoxtecTM, FOSS in North America, Eden Prairie, MN)

and is used for some quality control applications.

8.3.4.2 Preparation of Sample

If the sample contains more than 10% H2O, dry the

sample to constant weight at 95–100◦_{C under pressure}

≤100 mm Hg for about 5 h (AOAC Method 934.01).

8.3.4.3 Procedure (See Fig.8-2)

1. Weigh, to the nearest mg, about 2 g of predried

sample into a predried extraction thimble, with porosity permitting a rapid flow of ethyl ether. Cover sample in thimble with glass wool.

2. Weigh predried boiling flask.

3. Put anhydrous ether in boiling flask. Note: The

anhydrous ether is prepared by washing com-mercial ethyl ether with two or three portions

of H2O, adding NaOH or KOH, and letting

stand until most of H2O is absorbed from the

ether. Add small pieces of metallic Na and let hydrogen evolution cease (AOAC Method 920.39B). Petroleum ether may be used instead of anhydrous ether (AOAC Method 960.39).

4. Assemble boiling flask, Soxhlet flask, and

con-denser.

5. Extract in a Soxhlet extractor at a rate of five or

six drops per second by condensation for about 4 h, or for 16 h at a rate of two or three drops per second by heating solvent in boiling flask.

6. Dry boiling flask with extracted fat in an air

oven at 100◦_{C for 30 min, cool in desiccator, and}

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Soxhlet extraction apparatus.

8.3.4.4 Calculation

% Fat on dry weight basis

= (g of fat in sample/g of dried sample) ×100 [3]

8.3.5 Discontinuous Solvent Extraction Methods

8.3.5.1 Mojonnier Method

8.3.5.1.1 Principle and Characteristics Fat is extra-cted with a mixture of ethyl ether and petroleum ether in a Mojonnier flask, and the extracted fat is dried to a constant weight and expressed as percent fat by weight.

The Mojonnier test is an example of the discontin-uous solvent extraction method and does not require removal of moisture from the sample. It can be applied to both liquid and solid samples. If petroleum ether is used to purify the extracted fat, this method is very similar to the Roese-Gottlieb Method (AOAC Method 905.02) in both principle and practice. The Mojonnier

flasks (Fig.8-3) are used not only for the Mojonnier and

Roese-Gottlieb methods, but also to do the hydrolysis (acid, alkaline, or combination) prior to fat extraction and GC analysis to determine fat content and fatty acid

profile (Sect.8.3.6). (Note that sometimes the terms

Mojonnier, Roese Gottlieb, and alkaline hydrolysis are

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a.Mojonnier Method:

• Lemak diekstraksi dengan campuran pelarut etil eter dan petroleum eter dalam Labu Mojonnier.

• Dapat digunakan untuk sampel padat dan cair. • Roise-Gottlieb Method (AOAC No. 905.02).

• Labu Mojonnier digunakan juga untuk hidrolisis (basa atau asam), terhadap sampel, sebelum diekstrak.

3. Metode Ekstraksi Diskontinyu

Terdiri dari:

a.Mojonnier Method

b.Chloroform-Methanol Method.

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Mojonnier Method . . .

• Terutama untuk análisis kadar lemak pada dairy food (AOAC No. 989.05).

• Digunakan juga untuk analisis kadar lemak pada tepung (AOAC No. 922.06) dan pakan ternak (AOAC No. 954.02). • Sebelum ekstraksi, dilakukan hidrolisis (asam/HCl). • Prosedur: 10g sampel, masukan dalam labu,

tambahkan 1,5-2,0 ml NH4OH, kocok, tambahkan 10ml

etanol 95%, kocok, tambahkan 25ml etil eter, kocok, tambahkan 25ml petroleum eter, kocok, sentrifius, pidahkan larutan. lakukan tiga kali. larutan yang mengandung lemak dikeringkan dengan oven.

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Mojonnier Flask

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b.Chloroform-Methanol Method:

• Methanol untuk melarutkan komponen polar, sedangkan Chloroform untuk melarutkan lemak.

• Kemudian ditambahkan 0,88% KCl, untuk memisahkan fase polar dan non polar.

• Digunakan untuk ektraksi bahan rendah lemak. • Prosedur:

• 1 g sampel ditambahkan 10 ml methanol, homogenize 1min, tambah 20 ml chloroform, homogenize 1min, saring, cairan ditambah 0.88% HCl.

• Tebentuk 2 fase, dipisahkan. Fase chloroform

(mengandung lemak) di tambah aquadest (pencucian), pisahkan, tambahkan NaSO4, kemudian dikeringkan

hingga konstan.

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B. METODE EKSTRAKSI TANPA PELARUT

• AOAC No. 989.04 dan 989.10 (dairy products), AOAC No. 964.12 (seafood), dan AOAC No. 932.11 (flavor extract). • H2SO4 pekat ditambahkan pada sampel (dairy products),

dalam botol Babcock.

• Asam menguraikan protein, menghasilkan panas, melepaskan lemak.

• Lemak dipisahkan dengan penambahan air panas dan sentrifugasi.

• Volume lemak dapat diketahui langsung pada botol Babcock.

1. Babcock Method

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130 Part II • Compositional Analysis of Foods

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Babcock milk test bottles for milk (a), cream (b), and cheese (Paley bottle) (c) testing. (Cour-tesy of Kimble Glass Co., Vineland, NJ.)

8.4.1.3 Applications

The Babcock method, which is a common official method for the determination of fat in milk, takes about 45 min and duplicate tests should agree within 0.1%. The Babcock method does not determine the phospholipids in the milk products. It is not applicable to products containing chocolate or added sugar with-out modification because of charring of chocolate and sugars by sulfuric acid. A modified Babcock method is used to determine essential oil in flavor extracts (AOAC Method 932.11) and fat in seafood (AOAC Method 964.12).

8.4.2 Gerber Method for Milk Fat 8.4.2.1 Principle

The principle of the Gerber method is similar to that of the Babcock method, but it uses sulfuric acid and amyl alcohol. The sulfuric acid digests proteins and carbo-hydrates, releases fat, and maintains the fat in a liquid state by generating heat.

8.4.2.2 Procedure

1. Transfer 10 ml of H2SO4 at 15–21◦C into a

Gerber milk bottle.

2. Accurately measure milk sample (11 ml) into the Gerber bottle, using a Gerber pipette.

3. Add 1 ml of isoamyl alcohol to the bottle.

4. Tighten the stopper and mix by shaking the bottle.

5. Centrifuge the bottle for 4 min.

6. Place the bottle in a water bath at 60–63◦_{C for}

5 min and then read the fat content from the graduations on the bottle neck.

8.4.2.3 Applications

The Gerber method is comparable to the Babcock method but is simpler and faster and has wider appli-cation to a variety of dairy products (14). The isoamyl alcohol generally prevents the charring of sugar found with the regular Babcock method. This test is more popular in Europe than in America.

8.5 INSTRUMENTAL METHODS

Instrumental methods offer numerous attractive fea-tures compared with the previously described extrac-tion methods. In general, they are rapid, nonde-structive, and require minimal sample preparation and chemical consumption. However, the equipment can be expensive and measurements often require the establishment of calibration curves specific to vari-ous compositions. Despite these drawbacks, several of the following instrumental methods are very widely used in quality control as well as research and prod-uct development applications. The following section describes several of these instrumental methods. 8.5.1 Infrared Method

The infrared (IR) method is based on absorption of IR energy by fat at a wavelength of 5.73µm. The more the energy absorption at 5.73µm, the higher is the fat content of the sample (15). Mid-IR spectroscopy is used in Infrared Milk Analyzers to determine milk fat content (AOAC Method 972.16). Near-infrared (NIR) spectroscopy has been used to measure the fat content of commodities such as meats, cereals, and oilseeds in the laboratory and is being adapted for on-line measurement. See Chap. 23 for a discussion of IR spectroscopy.

8.5.2 Specific Gravity (Foss-Let Method) Fat content by the Foss-Let method (Foss North Amer-ica, Eden Prairie, MN) is determined as a function of the specific gravity of a sample solvent extract. A sample of known weight is extracted for 1.5–2 min in a vibration-reaction chamber with perchloroethy-lene. The extract is filtered, and using a thermostati-cally controlled device with digital readout, its specific Babcock Bottles Babcock Centrifuge

Analisis Lipid: Tanpa pelarut . . .

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• Metode análisis lemak untuk dairy products, dengan menggunakn pelarut Asam Sulfat dan Amyl Alcohol. • Menggunakan Gerber Bottle.

• As. Sulfat untuk mennguraikan protein dan kabohidrat, mengeluarkan panas, dan melepaskan lemak.

• Amyl Alcohol mencegah pengarangan gula (laktosa). • Banyak digunakan di Eropa.

• Volume lemak dapat diketahui langsung, seperti pada metode Babcock.

2. Gerber Method

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Gerber Bottles

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C. METODE INSTRUMENTASI

• Didasarkan peda penyerapan energi infra merah (panjang gelombang 5,73 um) oleh lemak.

• Semakin besar absorban (serapan sinar infra merah), semakin tinggi konsentrasi lemak dalam sampel. • Menggunakan alat: Specteometer Infra Merah. • Mid-IR untuk susu (AOAC No. 972.16).

• Near-IR untuk sereal, daging, dan bĳi-bĳian.

1. Infrared Method

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Analisis Lipid : Instrumentasi . . .

• Didasarkan pada specific gravity larutan yang mengandung lemak terekstrak.

• Sampel diekstrak dengan Perchloroethylene, disaring, larutan ditimbang.

• Berat lemat diketahui dengan membandingkan tabel atau grafik.

2. Specific Grafity Method

3. NMR Method

• Didasarkan pada kurva titik leleh lemak yang dikandung dalam bahan.

• Sangat cepat dan akurat, sangat mahal.

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Analisis Lipid : Instrumentasi . . .

4. Gas Chromatography Method

• Digunakan untuk menentukan jenis trigliserida dan asam lemak dalam lemak.

• Pemisahan jenis trigliserida dan asam lemak didasarkan pada beda afinitas pada kolor kromatografy.

• Total lemak merupakan jumlah dari semua asam lemak yang terdapat dalam bahan.

• Untuk mengetahui jenis asam lemak, diperlukan standar asam lemak.

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Chromatogram fatty acid analysis by GC

128 Part II • Compositional Analysis of Foods

Auto-Scaled Chromatogram pA 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 Minutes 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 Tetranoic Hexanoic

Octanoic DecanoicInternal

Standard Dodecanoic Tridecanoic Tetradecanoic 9c-Tetradecenoic Pentadecanoic _{10c-Pentadecenoic} Hexadecanoic 9t-Hexadecenoic 9c-Hexadecenoic Heptadecanoic _{10c-Heptadecenoic} Octadecanoic 9t-Octadecenoic 9c-Octadecenoic 9t,12t Octadecadienoic 9c, 12c Octadecadienoic Eicosanoic 6c,9c,12c Octadecatrienoic 11c-Eicosenoic 9c,12c,15c-Octadecatrienoic 6,9,12,15c Octadecatetraenoic 11c, 14c-Eicosadienoic Docosanoic 13c-Docosenoic 11c,14c,17c-Eicosatrienoic 5,8,11,14c-Eicosatetraenoic Tetracosanoic 5,8,11,14,17c Eicosapentaenoic 15c-Tetracosenoic 7,10,13,16,19cDocosapentaenoic 4,7,10,13,16,19Docosahexaenoic 8-5 f i g u r e

Example of chromatogram from gas chromatography analysis.

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table Fatty Acid Conversion Table

Molecular Weight Conversion Factorsb

Methyl 1/3 Triglyceride/ Acid/Methyl Acid/ Fatty Acida Acid Ester Triglyceride Triglyceride Methyl Ester Ester Triglyceride 4:0 Butyric 88.11 102.14 302.38 100.79 0.9868 0.8627 0.8742 Tetranoic 5:0 Valeric 102.40 116.43 345.25 115.08 0.9885 0.8795 0.8898 Pentanoic 6:0 Caproic 116.16 130.19 386.53 128.84 0.9897 0.8923 0.9016 Hexanoic 7:0 Heptanoic 130.19 144.22 428.62 142.87 0.9907 0.9027 0.9112 8:0 Caprylic 144.21 158.24 470.69 156.90 0.9915 0.9114 0.9192 Octanoic 10:0 Capric 172.27 186.30 554.85 184.95 0.9928 0.9247 0.9314 Decanoic 12:0 Lauric 200.35 214.38 639.02 213.01 0.9937 0.9346 0.9405 Dodecanoic 13:0 Tridecanoic 214.35 228.38 681.10 227.03 0.9941 0.9386 0.9441 (continued) 23 24