Lipids (Part-1) || Food Analysis || Pharmaceutical Analysis Department || M.Pharmacy (Sem-1)

1
FOOD ANALYSIS
2
LIPIDS VITAMINS
3
LIPIDS
Lipids are organic compounds that contain
hydrogen, carbon, and oxygen atoms, which forms
the framework for the structure and function of
living cells.
4
LIPIDS INTRODUCTION

• Lipids are one of the major constituents of
  foods, and are important in our diet for a number
  of reasons.
• They are a major source of energy and provide
  essential lipid nutrients.
• Nevertheless, over-consumption of certain lipid
  components can be detrimental to our health,
•  e.g. cholesterol and saturated fats.
• In many foods the lipid component plays a major
  role in determining the overall physical
  characteristics, such as flavor, texture, mouth
  feel and appearance.

5

• For this reason, it is difficult to develop
  low-fat alternatives of many foods, because once
  the fat is removed some of the most important
  physical characteristics are lost.
• Finally, many fats are prone to lipid oxidation,
  which leads to the formation of off-flavors and
  potentially harmful products.
• Some of the most important properties of concern
  to the food analyst are
• Total lipid concentration
• Type of lipids present
• Physicochemical properties of lipids, e.g., crysta
  llization, melting point, smoke point, rheology,
  density and color
• Structural organization of lipids within a food

6

• Properties of Lipids in Foods
• Lipids are usually defined as those components
  that are soluble in organic solvents (such as
  ether, hexane or chloroform), but are insoluble
  in water.
• This group of substances includes triacylglycercol
  s, 
• diacylglycercols, monoacylglycercols, free fatty
  acids, phospholipids, sterols, caretonoids and
  vitamins A and D.
• The lipid fraction of a fatty food therefore
  contains a complex mixture of different types of
  molecule.
• Even so, triacylglycercols are the major
  component of most foods, typically making up more
  than 95 to 99 of the total lipids present. 

7

• Triacylglycerols are esters of three fatty acids
  and a glycerol molecule.
• The fatty acids normally found in foods vary in
  chain length, degree of unsaturation and position
  on the glycerol molecule.
• Consequently, the triacylglycerol fraction itself
  consists of a complex mixture of different types
  of molecules.
• Each type of fat has a different profile of
  lipids present which determines the precise
  nature of its nutritional and physiochemical
  properties.
• The terms fat, oil and lipid are often used
  interchangeably by food scientists. Although
  sometimes the term fat is used to describe those
  lipids that are solid at the specified
  temperature, whereas the term oil is used to
  describe those lipids that are liquid at the
  specified temperature.

8
LIPIDS CLASSIFICATION

• They may be classified based on their physical
  properties at room temperature (solid or liquid,
  respectively fats and oils), on polarity, or on
  their essentiality for humans, but the preferable
  classification is based on their structure.
• Based on structure, they can be classified in
  three major groups.
• Simple lipids,
• Complex lipids,
• Derived lipids

9

• 1.Simple lipids
• They consist of two types of structural
  moieties.They include
• glyceryl esters that is esters of glycerol and
  fatty acids
• e.g. triacylglycerols, mono- and diacylglycerols
• cholesteryl esters that is esters of cholesterol
  and fatty acids
• waxes which are esters of long-chain alcohols and
  fatty acids, so including esters of vitamins A
  and D
• ceramides that is amides of fatty acids with
  long-chain di- or trihydroxy bases containing
  1222 carbon atoms in the carbon chain
• e.g. sphingosine.
•  

10

• 2.Complex lipidsThey consist of more than two
  types of structural moieties.They include
• phospholipids that is glycerol esters of fatty
  acidsphosphoric acid, and other groups
  containing nitrogen
• phosphatidic acid that is diacylglycerol
  esterified to phosphoric acid
• phosphatidylcholine that is phosphatidic acid
  linked to choline, also called lecithin
• phosphatidyl acylglycerol in which more than one
  glycerol molecule is esterified to phosphoric
  acid e.g. cardiolipin and diphosphatidyl
  acylglycerol
• glycoglycerolipids that is 1,2-diacylglycerol
  joined by a glycosidic linkage through position
  sn-3 with a carbohydrate moiety
• gangliosides that is glycolipids that are
  structurally similar to ceramide polyhexoside and
  also contain 1-3 sialic acid residues most
  contain an amino sugar in addition to the other
  sugars
• sphingolipids, derivatives of ceramides
• sphingomyelin that is ceramide phosphorylcholine

11

•  3.Derived lipids
• They occur as such or are released from the other
  two major groups because of hydrolysis that is
  are the building blocks for simple and complex
  lipids.They include
• fatty acids and alcohols
• fat soluble vitamins A, D, E and K
• hydrocarbons
• sterols.

12
LIPIDS GENERAL METHODS OF ANALYSIS OF LIPIDS
ANALYTICAL PARAMETERS FOR OILS AND FATS 

• The properties of oils and fats vary along with
  the degree of unsaturation, average molecular
  weight and also acidity from hydrolysis.
• A number of parameters are used for their
  analysis which are included under physical
  constants and chemical constants.
• Physical constants include viscosity, specific
  gravity, refractive index, solidification point
  etc.

13

• Following is a brief idea about some of the
  analytical parameters grouped under chemical
  constants.
• Iodine value,
• Saponification value,
• Acid value,
• Hydroxyl value,
• Acetyl value,
• Unsaponifiable matter,
• Peroxide value,
• Kreistest (rancidity index),
• Ester value,
• Reichert Messle Value ,
• Polenski value.

14

• Iodine value
• Definition
• Iodine value is the number, which express in
  grams, a quantity of halogen, calculated as
  iodine which is absorbed by 100g of the substance
  under the described condition. Iodine value may
  be determined by iodine monochloride method,
  iodine monobromide method, pyridine monobromide
  etc.
• Significance
• Iodine value is the measure of unsaturation (
  number of double bond ) in fat.
• Iodine number is useful to analyze the degree
  of adulteration
• On basis of iodine value the oils can be
  differentiated into non-drying oil and semidrying
  oil. Drying oil shows less iodine value,
  non-drying oil shows more iodine value and
  semidrying oil shows moderate iodine value.

15
Procedure Iodine Monochloride Method place an
accurately weighed quantity of substance being
examined(castor oil) in a dry 250ml capacity
iodine flask. Add 1ml of carbon tetrachloride
and dissolve in 20ml of iodine monochloride
solution. Insert the stopper and allow to stand
in the dark at a temperature in between 15-25
degrees Celsius for 30 mins. Place 15ml of
potassium iodide solution and cup top, carefully
remove the stopper, rinse the stopper and sides
of the flask with 10ml of water, shake and
titrate with 0.1M sodium thiosulphate using
starch as an indicator. The starch solution
added towards the end of the titration. Note the
ml required (a). Repeat the operation omitting
the substance being examined and note the number
of ml required (b). calculate the iodide value
with the following expression. Iodine value
1.269(b-a)/w W Weight in grams of the
substance of the oil.
16

• 2. Saponification value
• It is defined as the number of milligrams of KOH
  required to neutralize the fatty acids resulting
  from complete hydrolysis of 1 gm of the sample of
  oil or fat.
• Significance
• Saponification value of fat or oil is one of its
  characteristic physical properties.
• Saponification value occurs in an inverse
  proportion to the average molecular weight of
  fatty acid present in oil.
• Higher saponification number for fats
  containing short chain fatty acids.
• Saponification value
• This value is normally applied for butter fat,
  coconut oil in which lower fatty acids glycerides
  occur in high content. It is used for detecting
  adulteration Saponification value is determined
  by refluxing a known amount of sample with excess
  of standard alcoholic KOH

17
Procedure 2 gm of the given sample of oil is
accurately weighed in a RB flask and refluxed
with 25ml of 0.5M ethanolic potassium hydroxide
with a little pumic powder in a water bath for 30
minutes. Add 1ml of phenolphthalein solution and
titrate immediately with 0.5M hydrochloric acid
(a ml) Carry out the blank, omitting the
substance under examination (b ml) Calculate the
saponification value Saponification value
28.05 (b- a)/w b volume of hydrochloric acid
consumed in blank titration a volume of
hydrochloric acid consumed in sample titration w
weight of the sample
18
3. Acid value It is defined as the number of
milligrams of potassium hydroxide required to
neutralize the free fatty acids present in 1gm of
sample of fat or oil. Significance Acid value
is used as an indication of rancid state.
Generally rancidity causes free fatty acids,
which have been liberated by hydrolysis of
glycerides due to the action of moisture,
temperature or enzyme lipase. Acid value Acid
value can be determined by treating sample with
solution of KOH using phenolphthalein as
indicator
19
Procedure Accurately weigh about 1gm of the oil
and to this add 50 ml mixture of equal volume of
ethanol (95) and ether, previously neutralized
with 0.1M of KOH to phenolphthalein solution.
Add 0.1 ml of phenolphthalein solution and
titrate with 0.1M KOH until the solution remains
faintly pink n number of milligrams of
potassium hydroxide required w- weight of the
sample. The STD for edible fats and oils
indicate that the acid value must not exceed 0.6
. The acid value can be determined by the
formulae Acid value 5.61
20
4. Hydroxyl value It is defined as number of
milligrams of potassium hydroxide required to
neutralize the acetic acid capable of combining
by acetylation with 1 g sample of fat or oil.
5. Acetyl value It is the number of
milligrams of potassium hydroxide required to
neutralize acetic acid obtained when 1g of sample
acetylated oil is saponified. Significance
Acetyl number is a measure of number of
hydroxyl groups present. to detect adulteration
and rancidity.
21
6. Unsaponifiable Matter It is the matter
present in fats and oil, which after
saponification by caustic alkali and subsequent
extraction with an organic solvent, remains
non-volatile on drying at 8oC. It includes
sterols (phytosterol and cholesterol), oil
soluble vitamins, hydrocarbons and higher
alcohols. Paraffin hydrocarbons can be detected
by this method as adulterants. 7. Peroxide
Value Peroxide Value Is the number which
expresses in milli equivalents of active oxygen
that expresses the amount of peroxide containing
1000gms (kg) of substances (meq/kg). It is a
measure of peroxides present in oil. Aperoxide
value is generally less than 10 mEqkg in fresh
samples of oil. Due to temperature or storage,
rancidity occurs causing increase in peroxide
values.
22
8.Kreistest (rancidity index) Due to rancidity,
epihydrin aldehyde or malonaldehyde are increased
which are detected by Kreis test using
phloroglucinol which produces red colour with the
oxidized fat. 9.Ester value It is defined as
number of milligrams of potassium hydroxide
required to combine with fatty acids which are
present in glyceride form in 1 g sample of oil or
fat. Difference between saponification value and
acid value is ester value..
23
10. Reichert messle value This value is a
measure of volatile water soluble acid contents
the fat. It is defined as number of milli litres
N/10 potassium hydroxide solution required to
neutralize the volatile water soluble fatty acids
obtained by 5 g fat. Significance Higher
content of volatile fatty acids of butter
responsible for its higher reichert-meissl
number. It is useful in testing
purity/adulteration of butter. 11.Polenski
Value It is defined as the number of millitres
of N/10 potassium hydroxide solution required to
neutralize water-insoluble, steam - distillable
acids liberated by hydrolysis of 5 gm of fat.
Significance The Polenski value is an
indicator of how much volatile fatty acid can be
extracted from fat through saponification.
24
LIPID CONTENT ANALYSIS 

• Gravimetric Method
• (1) Wet extraction Roese Gottliegb
  Mojonnier.
• (2) Dry extraction Soxhlet Method.
• 2. Volumetric Methods (Babcock, Gerber Methods)
  

25

• Gravimetric Method
• (1) Wet Extraction Roese Gottlieb
  Mojonnier.
• For Milk
• 1) 10 g milk 1.25 ml NH4OH mix. Solubilizes
  protein and neutralizes.
• 2) 10 ml EtOH shake. Begins extraction,
  prevents gelation of proteins.
• 3) 25 ml Et2O shake and mix.
• 4) 25 ml petroleum ether, mix and shake.

26
(2) Dry Extraction Soxhlet Method. Sample in
thimble is continuously extracted with ether
using Soxhlet condenser. After extraction,
Direct measurement of fat evaporate ether and
weigh the flask. Indirect measurement dry
thimble and weigh thimble and sample.
27

• 2. Volumetric Method (Babcock, Gerber Methods)
• Theory
• Treat sample with H2SO4 or detergent.
• Centrifuge to separate fat layer.
• Measure the fat content using specially
  calibrated bottles.
• Methods
• Known weight sample.
• H2SO4 digest protein, liquefy fat.
• Add H2O so that fat will be in graduated part of
  bottle.
• centrifuge to separate fat from other materials
  completely.

28
LIPIDS REFINING OF FATS AND OILS
Objectives of Refining 1- In refining, physical
and chemical processes are combined to remove
undesirable natural as well as environmental-relat
ed components from the crude oil. 2-These
components comprise for example phosphatides,
free fatty acids, pigments (such as chlorophyll),
odors and flavors (including aliphatic aldehyde
and ketone), waxes as well as heavy metals,
pesticides etc. 3-Removal of undesired products
from crude oils ? free fatty acids (FFA) ?
phospholipids (gums) ? oxidised products ? metal
ions ? colour pigments ? other impurities 4-
Preservation of valuable vitamines. (vitamina E
ortocopherolnatural anti-oxidants) 5- Minimize
oil losses 6- Protection of the oil against
degradation
29

• Methods of Refining
• Chemical Refining
• The Chemical Refining process is used for oils
  and fats with low FFA and contains three basic
  steps
• ? Neutralizing
• ? Bleaching
• ? Deodorizing
• Residual soap and gums removal in neutralizing is
  accomplished by either water washing or using a
  silica adsorbent in bleaching.
• Physical RefiningThe Physical Refining process
  is used for oils and fats with high FFA and
  contains three basic steps
• ? Acid Conditioning or Enhanced Degumming
• ? Bleaching
• ? Stripping and Deodorizing
• The degumming process used depends on the oil or
  fat being refined. 

30
Depending on the requirements, the following
basic processes are implemented ? degumming for
removal of phosphatides, ? neutralization for
removal of free fatty acids, ? bleaching for
removal of color, ? deodorization to distill
odors and flavors as well as free fatty acids and
? winterization for separation of waxes.
31

• 1.Chemical Refining
• Neutralizing
• Objective Removal of free fatty acids
• Batch Neutralization Refining of vegetable oils
  is essential to ensure removal of gums, waxes,
  phosphatides and free fatty acid (F. F.A.) from
  the oil to impart uniform colour by removal of
  colouring pigments and to get rid of unpleasant
  smell from the oil by removal of odiferous
  matter.
• Refining is carried out either on batch operation
  or as continuous operation. With certain oils
  even physical refining can be carried out instead
  of chemical. For processing less than thirty
  tones of oil per 24 hours, and when oil has F.F
  .A. content of 1 or less normally batch process
  is recommended. Batch process involves low
  capital investment, simplicity of operation and
  low maintenance, making refining economically a
  viable proposition even at capacity as low as 10
  tonnes per 24 hours. 

32

• Bleaching
• Objective bleaching for removal of color,
• ? Silica adsorption reduces water consumption,
  effluent treatment and bleaching earth
  consumption
• ? Pre-bleaching oil minimizes bleaching earth
  consumption
• Deodorizing
• With a light color. Deodorization consists of
  steam sparging the oil under high vacuum (lt 10 mm
  Hg) at high temperatures (gt 200C). After
  deodorization and cooling of the oil, a chelating
  agent, such as citric acid, may be added to
  deactivate trace metals. Antioxidants may also be
  added to enhance stability. 

33
2.Physical Refining Acid Conditioning or
Enhanced Degumming Degumming Objective
degumming for removal of phosphatides, The aim
of degumming operation ? The emulsifying action
of phospholipids increases oil losses during
alkali refining. ? Gums lead brown discoloration
of oil after heating during deodorization. ?
Salts may be formed with cooper,
magnesium,calcium and iron, accelerating
oxidative degredation of oil. Certain
phospholipids, such as lecithin, find widespread
industrial application. Different degumming
processes are carried out to remove phosphatides.
For efficient and economic application of this
procedure appropriate machines and equipments are
used. 1.Water degumming 2.Acid degumming
3.Enzymatic degumming 4.Membrane degumming .
34

• Acid degumming
• There are two type of Acid degumming
• 1- Dry acid degumming
• 2- Wet acid degumming
• Acid Degumming Process Steps
• ? Heat oil to 60 -70 C
• ? Acid addition and mixing
• ? Hydration mixing 30 minutes
• ? Centrifugal separation of hydrated gums
• ? Vacuum drying of degummedoil
• ? Gums -recombined in meal 

35

• Water degumming
• Water Degumming Process Steps
• Heat oil to 60 -70 C
• Water addition and mixing
• Hydration mixing 30 minutes
• Centrifugal separation of hydrated gums
• Vacuum drying of degummed oil
• Gums -dried for edible lecithin or recombined in
  meal 

36

• Enzymatic degumming
• Enzymatic degumming was first introduced by the
  German Lurgi Company as the Enzy Max process
  .The EnzyMax process can be divided into four
  different steps
• the adjustment of the optimal conditions for the
  enzyme reaction, i.e. optimal pH with a citrate
  buffer and the optimal temperature
• the addition of the enzyme solution
• the enzyme reaction
• the separation of lysophosphatide from the oil at
  about 75 C.
• Enzymes for enzymatic degumming
• ? Lecitase 10L (pancreatic phospholipase A2)
• ? Lecitase Novo (microbial lipase)
• ? Lecitase Ultra (microbial lipase)

37
Thank you