lipid analysis copy - unila
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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➡ 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 dari asam
lemak C2-C24, dihitung sebagai trigliserida. ➡ Lipid Classification:
• Lipid sederhana (Simple lipid), • Lipid komplek (Compound lipid),
• Lipid Turunan (Derivate lipid).
Analisis Lipid . . .
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Analisis Lipid: Pendahuluan . . .
➡ 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 tidak sesuai dengan standar dan menyebabkan kerugian yang besar
➡ 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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Analisis Lipid . . .
M E T O D E A N A L I S I SA. Metode Ekstraksi Pelarut:
1. Metode Ekstraksi Kontinyu (Goldfish Method). 2. Metode Ekstraksi Semi Kontinyu (Soxhlet Method). 3. Metode Ekstraksi Diskontinyu (Mojonnier 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.
➡ Persiapan Sampel:
• Mempengaruhi validitas pengujian. • Disesuaikan dengan jenis sampel. • Tahapan persiapan sampel:
a. Pengeringan. b. Pengecilan ukuran.
c. Hidrolisis �5
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Analisis Lipid : Ekstraksi pelarut . . .
➡ 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 murni dan bebas peroksida. • Rasio pelarut dan bahan terlarut penting,
memaksimalkan ekstraksi.
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Analisis Lipid : Ekstraksi pelarut . . .
• 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 “channel ing”,
mengebabkan esktraksi tidak sempurna.
1. Metode Ekstraksi Kontinyu (Goldfish Method)7
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Analisis Lipid . . .
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Goldfish Fat Extractor8
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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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Analisis Lipid . . .
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Soxhlet Fat Extractor
Chapter 8 • Fat Analysis 123
8.3.4 Semicontinuous Solvent ExtractionMethod: Soxhlet Method
The Soxhlet method (AOAC Method 920.39C forCereal Fat; AOAC Method 960.39 for Meat Fat) (8) isan example of the semicontinuous extraction methodand is described below.
8.3.4.1 Principle and Characteristics
For semicontinuous solvent extraction, the solventbuilds up in the extraction chamber for 5–10 min andcompletely surrounds the sample and then siphonsback to the boiling flask. Fat content is measured byweight loss of the sample or by weight of the fatremoved.
This method provides a soaking effect of thesample and does not cause channeling. However,this method requires more time than the continuousmethod. 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 thesample 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 predriedsample into a predried extraction thimble, withporosity 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 portionsof H2O, adding NaOH or KOH, and lettingstand until most of H2O is absorbed from theether. Add small pieces of metallic Na andlet hydrogen evolution cease (AOAC Method920.39B). Petroleum ether may be used insteadof 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 orsix drops per second by condensation for about4 h, or for 16 h at a rate of two or three drops persecond by heating solvent in boiling flask.
6. Dry boiling flask with extracted fat in an airoven at 100◦C for 30 min, cool in desiccator, andweigh.
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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 ExtractionMethods
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 etherin a Mojonnier flask, and the extracted fat is driedto a constant weight and expressed as percent fat byweight.
The Mojonnier test is an example of the discontin-uous solvent extraction method and does not requireremoval of moisture from the sample. It can be appliedto both liquid and solid samples. If petroleum ether isused to purify the extracted fat, this method is verysimilar to the Roese-Gottlieb Method (AOAC Method905.02) in both principle and practice. The Mojonnierflasks (Fig. 8-3) are used not only for the Mojonnier andRoese-Gottlieb methods, but also to do the hydrolysis(acid, alkaline, or combination) prior to fat extractionand GC analysis to determine fat content and fatty acidprofile (Sect. 8.3.6). (Note that sometimes the termsMojonnier, Roese Gottlieb, and alkaline hydrolysis areused interchangeably.)
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Analisis Lipid . . .
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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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Analisis Lipid . . .
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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 13
Analisis Lipid . . .
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Mojonnier Flask Shaker
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Analisis Lipid . . .
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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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Analisis Lipid . . .
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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 officialmethod for the determination of fat in milk, takesabout 45 min and duplicate tests should agree within0.1%. The Babcock method does not determine thephospholipids in the milk products. It is not applicableto products containing chocolate or added sugar with-out modification because of charring of chocolate andsugars by sulfuric acid. A modified Babcock methodis used to determine essential oil in flavor extracts(AOAC Method 932.11) and fat in seafood (AOACMethod 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 ofthe Babcock method, but it uses sulfuric acid and amylalcohol. The sulfuric acid digests proteins and carbo-hydrates, releases fat, and maintains the fat in a liquidstate by generating heat.
8.4.2.2 Procedure
1. Transfer 10 ml of H2SO4 at 15–21◦C into aGerber milk bottle.
2. Accurately measure milk sample (11 ml) intothe Gerber bottle, using a Gerber pipette.
3. Add 1 ml of isoamyl alcohol to the bottle.
4. Tighten the stopper and mix by shaking thebottle.
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 thegraduations on the bottle neck.
8.4.2.3 Applications
The Gerber method is comparable to the Babcockmethod but is simpler and faster and has wider appli-cation to a variety of dairy products (14). The isoamylalcohol generally prevents the charring of sugar foundwith the regular Babcock method. This test is morepopular 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 preparationand chemical consumption. However, the equipmentcan be expensive and measurements often require theestablishment of calibration curves specific to vari-ous compositions. Despite these drawbacks, several ofthe following instrumental methods are very widelyused in quality control as well as research and prod-uct development applications. The following sectiondescribes several of these instrumental methods.
8.5.1 Infrared Method
The infrared (IR) method is based on absorption of IRenergy by fat at a wavelength of 5.73 µm. The morethe energy absorption at 5.73 µm, the higher is thefat content of the sample (15). Mid-IR spectroscopy isused in Infrared Milk Analyzers to determine milk fatcontent (AOAC Method 972.16). Near-infrared (NIR)spectroscopy has been used to measure the fat contentof commodities such as meats, cereals, and oilseedsin the laboratory and is being adapted for on-linemeasurement. See Chap. 23 for a discussion of IRspectroscopy.
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 functionof the specific gravity of a sample solvent extract.A sample of known weight is extracted for 1.5–2 minin 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 CentrifugeAnalisis Lipid: Tanpa pelarut . . . 17
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Analisis Lipid: Tanpa pelarut . . .
• 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 Method18
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Gerber BottlesAnalisis Lipid: Tanpa pelarut . . . 19
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Analisis Lipid . . .
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: Spectrometer 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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Analisis Lipid . . . Chromatogram fatty acid analysis by GC128 Part II • Compositional Analysis of Foods
Auto-Scaled Chromatogram
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8-5f igure
Example of chromatogram from gas chromatography analysis.
8-4table Fatty Acid Conversion Table
Molecular Weight Conversion Factorsb
Methyl 1/3 Triglyceride/ Acid/Methyl Acid/Fatty Acid a Acid Ester Triglyceride Triglyceride Methyl Ester Ester Triglyceride
4:0 Butyric 88.11 102.14 302.38 100.79 0.9868 0.8627 0.8742Tetranoic
5:0 Valeric 102.40 116.43 345.25 115.08 0.9885 0.8795 0.8898Pentanoic
6:0 Caproic 116.16 130.19 386.53 128.84 0.9897 0.8923 0.9016Hexanoic
7:0 Heptanoic 130.19 144.22 428.62 142.87 0.9907 0.9027 0.91128:0 Caprylic 144.21 158.24 470.69 156.90 0.9915 0.9114 0.9192
Octanoic10:0 Capric 172.27 186.30 554.85 184.95 0.9928 0.9247 0.9314
Decanoic12:0 Lauric 200.35 214.38 639.02 213.01 0.9937 0.9346 0.9405
Dodecanoic13:0 Tridecanoic 214.35 228.38 681.10 227.03 0.9941 0.9386 0.9441
(continued)
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