Lipid Analysis

Melisa Intan Barliana

Definition
Lipid together with proteins and carbohydrates,

are the principal structural components of foods.

Lipids are a group of substances that, in

general, are soluble in either, chloroform, or other organic solvents but are sparingly soluble in water.

Introduction
Among the most biologically significant properties of lipids are their hydrophobic properties. These properties are mainly due to a particular component of lipids: fatty acids, or simply fats. Fatty acids consist of an alkyl chain with a terminal carboxyl group.

Introduction
LIPID : a long chain ester from carboxylic acid ester and alcohol (ex: gliserol)
O OH2C H HO C O HC H HO C O OH2C H HO C C17H35 C17H35 C17H35

O H2C HC H2C O C O O O C O C C17H35 C17H35 C17H35

Gliserol

Asam stearat

Tristearin

Lipid Classification
This lipid classification based on lipid structure. 1. Fatty acyls The fatty acyl group in the fatty acids and conjugates class is characterized by a repeating series of methylene groups that impart hydrophobic character to this category of lipids.

2. Glycerolipids The glycerolipids essentially encompass all glycerol-containing lipids.

Monoacylglycerol

Diacylglycerol (DAG)

2. Glycerolipids The glycerolipids essentially encompass all glycerol-containing lipids.

Diacylglycerolphosphocholines

3. Sphingolipids Sphingolipids are a complex family of compounds that share a common structural feature, a sphingoid base backbone that is synthesized de novo from serine and a long chain fatty acyl-CoA, then converted into ceramides, phosphosphingolipids, glycosphingolipids, and other species, including protein adducts.

Sphinganine

5. Sterol lipids The sterol category is subdivided primarily on the basis of biological function.

Cholesterol

-estradiol

6. Prenol lipids Prenols are synthesized from the five carbon precursors isopentenyl diphosphate and dimethylallyl diphosphate that are produced mainly via the mevalonic acid pathway, also called isoprenoid.

-carotene

7. Saccharolipids Saccharolipids are compounds in which fatty acids are linked directly to a sugar backbone, forming structures that are compatible with membrane bilayers.

Monoacylaminosugars

8. Polyketides Polyketides are syntesized by polyketide synthases generate a much greater diversity of natural product structures, many of which have the character of lipids.

Aromatic polyketides: griseorhodin A

Saturation of Fatty Acids

Saturated fatty acid : fatty acid contain no double bonds between the individual carbon of fatty acid chain. Unsaturated fatty acid : fatty acid contain one or more double bonds, that is mono- or polyunsaturated (PUFA).

Saturated fatty acid

Contain only single CC bonds

Closely packed
Strong attractions between chains High melting points

Solids at room temperature also called fats

Saturated fatty acid

Butyric acid with 4 carbon atoms (contained in butter) Lauric acid with 12 carbon atoms (contained in coconut oil, palm oil, and breast milk) Myristic acid with 14 carbon atoms (contained in cow's milk and dairy products) Palmitic acid with 16 carbon atoms (contained in palm oil and meat) Stearic acid with 18 carbon atoms (also contained in meat and cocoa butter)

Unsaturated fatty acid

Contain one or more double C=C bonds Nonlinear chains do not allow molecules to pack

closely Few interactions between chains Low melting points Liquids at room temperature also called oils

Unsaturated fatty acid There are 3 types of unsaturated fatty acid, according to its unsaturation : Monounsaturated fatty acid are fatty acids that have one double bond in the fatty acid chain and all of the remainder of the carbon atoms in the chain are singlebonded. Polyunsaturated fatty acid (PUFA) are fatty acids that have more than one double bond in the fatty acid chain. Eicosanoid are eicosapolyenoat fatty acid derivatives, prostanoid (prostaglandin, prostacyclin, and thromboxane) ansd leucotriene.

Unsaturated fatty acid

Unsaturated fatty acid by its structure (Methylene-Interrupted Polyenes): -3 : -Linolenic acid (ALA) C18:3 (n-3) 9, 12, 15 (canola, soybean, walnut) Eicosapentaenoic acid (EPA) 20:5 (n3) (fish oil: salmon,cod, sardine) Docosahexaenoic acid (DHA) 22:6 (n-3) (fish oils) -6 : Linoleic acid (LA) C18:2 (n-6) (sun flower, corn oil) Arachidonic acid (AA) C20:4 (n-6) (peanut oil) -9 : Oleic acid C18:1 (n-9) (olive oil)

Oleic acid

Importance of Analysis
An accurate and precise quantitative analysis of lipids in foods is important for : Nutritional labeling, to determine whether the food meets the standard of identity and is uniform, and to understand the effects of fats and oils on the functional and nutritional properties of foods. Measurements of lipid stability impact not only the shelf life of the product but also its safety, since some oxidation products (e.g., malonaldehyde, cholesterol oxides) have toxic properties.

Lipid Function
Major source of energy for body

Hydrophobic barrier that permits partitioning of the aqueous contents of cells and subcellular structures.
Lipid as fat depot in mammalian and plant cells.

Lipid has additional function in body, such as coenzyme, control body`s homeostasis.

Lipid Analysis
Lipids are soluble in organic solvents and insoluble in water. Therefore, water insolubility is the essential analytical property used as the basis for the separation of lipids from proteins, water, and carbohydrates in foods and also for lipid analysis.
For lipid characterization, extraction of fat or oil from foodstuffs can be accomplished by homogenizing with a solvent combination such as hexane : isopropanol (3:2; vol/vol) or chloroform : methanol (2:1; vol/vol).

Lipid Analysis
based on physicochemicals properties of fat/oils 1. Melting Point
Fats being solid at room temperature, have a melting point that is higher than room temperature. Oils on the other hand have a melting point that is lower than room temperature. Those lipids that are neither solid fat nor liquid oil, but are semi-solid, have a melting point that is close to room temperature.
Principle Fat/oils put in the cappilary tube, chilled and heated gardually. The capillary tube melting point, also known as the complete melting point or clear point, is the temperature at which fat heated at a given rate becomes completely clear and liquid in a one-end closed capillary. Instrument : Thermometer

Clear point. A small amount of fat is placed in a capillary tube and heated at a controlled rate. The temperature at which the fat completely melts and becomes transparent is called the "clear point".
Slip point. A small amount of fat is placed in a capillary tube and heated at a controlled rate. The temperature at which the fat just starts to move downwards due to its weight is called the "slip point". Wiley melting point. A disc of fat is suspended in an alcohol-water mixture of similar density and is then heated at a controlled rate. The temperature at which the disc changes shape to a sphere is called the "Wiley melting point".

Lipid Analysis
2. Density measurement Comparition between volume of oil sample and water weigh at the same volume at certain point temperature (25C).

Instrument: Picnometer

Lipid Analysis
3. Turbidity Point or Crismer`s and Valenta test, is temperature where oil or fat change into solid phase. This measurement to determine pollutant by contaminant or foreign material Procedure: Oil sample put into measuring cup together with acetic acid or alcohol, heated until oil were dissolved completely (clear solution), chilled the solution gradually until it crystallized.

Temperature where soft fat crystal material were seen, recorded and determine as turbidity point or critical point.

Lipid Analysis
4. Refractive Index The refractive index (RI) of an oil is defined as the ratio of the speed of light in air (technically, a vacuum) to the speed of light in the oil.

RI is used to control hydrogenation; RI decreases linearly as iodine value decreases. RI also is used as a measure of purity and means of identification, since each substance has a characteristic RI.
Instrument: Refractometer

Lipid Analysis
5. Rancidity test : Kreis Test Principle: Fat will be oxidized (red) after adding phloroglucinol under acid condition.
Redness proportional to increase amount of epihydrine aldehyde product or malonaldehide as lipid oxidation product.

Lipid Analysis
6. Thibarbituric Acid (TBA) test

TBA test measures a secondary product of lipid oxidation, malonaldehyde.

TBA reacts with malonaldehyde (or malonaldehydetype products) to yield a colored compound that is measured spectrophotometrically.

Because the reaction is not specific to malonaldehyde, results sometimes are reported as TBA reactive substances (TBARS).

Lipid Analysis
7. Peroxide Value
1st Method
The fat or oil sample is dissolved in glacial acetic acid-isooctane (3:2). Upon addition of excess KI which reacts with the peroxides, iodine (I2) is liberated . The solution then is titrated with standardized sodium thiosulfate using a starch indicator.
ROOH + K+ II2 + Starch + 2Na2S2O3
Blue
H+, Heat

ROH + K+ OH- + I2 2NaI + Starch + Na2S4O6

Colorless

Procedure : Weigh oil sample, add 30 ml solvent, add 0.5 N saturated KI, put in dark room for 2 minutes while stirring, add 30 ml aquadest. Excessive Iod titrated with 0.1 N sodium thiosulfate.

7. Peroxide Value
2nd Method (Peroxide value determination using Colorimetry) Determine lipid hydroperoxide Hydroperoxide reacted with KI in acidity condition, liberated I2 determine by colorimetry. Liberated Iod determine at 560 nm after adding starch in HCL 0,1 N solution.

Lipid Analysis
8. Iodine Value Iodine value is defined as the grams of iodine absorbed per 100g sample. The iodine value (or iodine number) is a measure of degree of unsaturation, i.e., the number of carboncarbon double bonds in relation to the amount of fat or oil. There are 2 method: Hanus method Wijs method

Iodine Value

Principle: Unsaturated glyceride fat or oil has ability to absorb amount of iod.
The higher the amount of unsaturation, the more iodine is absorbed; therefore, the higher the iodine value, the greater the degree of unsaturation. Excessive Iod added to fat/oil, excessive iod were titrated with Na-thiosulfate.

Iodine Value
Hanus Method

Reagent : - 13,2 g Iod in 1 L acetic acid glacial until homogen, if added Brom (2ml) after the iod solution homogen and cold - Chloroform - KI solution 15% - Thiosulfate solution 0,1 N - Starch solution (indicator)

Iodine Value

Wijs method Reagent : 1. Chloroform or Carbon tetrachloride 2. Thiosulfate solution 0,1 N 3. KI solution 15 % 4. Starch solution (indicator) 5. Wijs reagent : Sublimized 13 Gram Iod added into 1L acetic acid glacial.

Lipid Analysis
9. Saponification Value
The saponification value (or saponification number) is defined as the amount of alkali necessary to saponify a given quantity of fat or oil. It is expressed as the milligrams of KOH required to saponify 1 g of the sample.
Saponification is the process of breaking down or degrading a neutral fat into glycerol and fatty acids by treatment of the fat with alkali

9. Saponification Value
Reagent: HCl 0.5 N Phenolftalein indicator Alcoholized KOH Prosedur : Weigh 5 g of oil/fat Add 50 mL alcoholized KOH

Hubungkan erlenmeyer yang telah berisi contoh dan KOH berlakohol dengan pendingin tegak. Refluks dengan menggunakan hot plate sampai semua contoh tersabunkan sempurna, yaitu sampai larutan bebas dari butiran lemak. Biasanya butuh 1 jam. Larutan didinginkan dan bagian dalam pendingin tegak dibilas dengan aquadest. Tambahkan 1 ml Indikator fenolftalein Titrasi dengan HCl 0.5 N sampai warna merah jambu hilang Lakukan penetapan Blanko.
Perhitungan : Bilangan Penyabunan : Titer Blanko Titer sampel x N HCl x 56.1 Berat Contoh

Lipid Analysis
10. Acid Value Acid value express as miligram of KOH require to neutralized free fatty acid in 1 gram oil/fat. Acid value is defined as amount of free fatty acid in oil/fat, usually related with oil/fat hydrolysis process to its quality.

10. Acid Value

Pereaksi

KOH 0,1 N Indikator Fenolftalein 1 % Alkohol 95% netral Prosedur : Timbang 20 Gram minyak/lemak Tambahkan 50 ml alkohol 95% netral, panaskan sampai mendidih Larutan dititrasi dengan KOH 0,1 N menggunakan indikator pp sampai terbentuk warna merah jambu yang persisten 10 detik

10. Acid Value Perhitungan Bilangan asam =

ml KOH x N KOH x 56.1 Berat sampel ml KOH x N KOH x M 10 G

Kadar asam =

G = berat sampel M = berat molekul asam lemak yang dominan dalam minyak/lemak. Untuk minyak kelapa = 205, minyak kelapa sawit = 263 dan asam oleat : 282.

1. Tuliskan struktur dari asam lemak (fatty acid) 2. Lipid apa saja yang memiliki fungsi sebagai pembentuk struktur membran sel 3. Sebutkan prinsip dari TBA test 4. Sebutkan prinsip dari uji ketengikan 5. Sebutkan perbedaan asam lemak jenuh dan tak jenuh