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Structure of the Major Phospholipids

0005 The chemical backbone of the major phospholipids is a

diacylglycerol molecule with the third carbon attached

to a phosphate molecule. Choline, ethanolamine,

serine, and inositol can be attached to the phosphate

group to change the physical and functional proper-

ties, leading to the formation of phosphatidylcholine,

phosphatidylethanolamine, phosphatidylserine, and

phosphatidylinositol, respectively. The groups at-

tached to positions 1 and 2 (a or b) are C

–C

fatty

acids with double bonds associated with the lecithin

source. The second carbon of the glycerol molecule,

the b-position, usually contains linoleic acid.

Physical Properties

0006 There are two major physical classes of lecithins: (1)

fluid, and (2) waxy solids.

0007 The fluid lecithins can have viscosities from 5000

to 100 000 cP, depending on processing conditions

and diluents. The low-viscosity products are made

through the addition of fatty acids and vegetable oil,

depending on the function and stability required. Di-

valent metal ions like calcium can be added during

drying to decrease viscosity. The moisture content can

also make a difference. Water levels above 1% will

increase the viscosity, eventually to a plastic state.

0008Deoiled lecithins are waxy solids that can be

ground to various particle sizes. They are stable,

free-flowing granules or powders.

Functional Properties

0009Lecithins are multifunctional agents. They can be

used for many purposes in a food system, as shown

in Table 2. The most popular functionalities are

discussed below.

0010Antidusting agents Lecithins reduce static electri-

city by wetting dusty particles. They can be used

alone or in conjunction with vegetable oils. Oils can

be selected for the degree of shelf-life required.

0011Crystal formation modifier Lecithins retard nucle-

ation in fats and even monoglycerides, reducing

graininess in texture.

0012Emulsifiers Lecithins are most often used as ampho-

teric emulsifiers. They promote stable formation of

oil-in-water and water-in-oil emulsions by reducing

tbl0001 Table 1 Chemical composition of soya bean lecithin

Granularlecithin Typicalliquidlecithin

Phosphatides (acetone-insolubles) (%) 95 (minimum) 60 (minimum)

Soya bean oil (%) 2–3 39

Moisture (%) 1 0.7

Fat (g per 100 g product) 90 93

Monounsaturated (oleic acid) (%) 9.2 17.9

Polyunsaturated (linoleic, linolenic acids) (%) 65.9 60.7

Saturated (palmitic, stearic acids) (%) 24.9 20.3

Carbohydrates (g per 100 g of product) 8 5

Approximate composition (100 -g sample)

Fatty acid content (g) 50 66

Fatty acid content (relative composition) (%)

Linoleic 58.9 54.0

Linolenic 7.0 6.7

Oleic 9.2 17.9

Palmitic 20.3 15.6

Stearic 4.6 4.7

Other fatty acids 0.0 1.1

Total 100.0 100.0

Primary acetone insolubles (g)

Phosphatidylcholine 23 15

Phosphatidylethanolamine 20 12

Phosphatidylinositol 14 9

Elemental analysis (mg)

Calcium 65 40

Iron 2 1

Magnesium 90 60

Phosphorus 3000 2000

Potassium 800 440

Sodium 30 10

Reproduced from Central Soya, Chemurgy Division (1989) The Lecithin Book. Fort Wayne: Central Soya.

PHOSPHOLIPIDS/Properties and Occurrence 4515

the interfacial surface tension between immiscible

liquids. (See Emulsifiers: Organic Emulsifiers.)

0013 Mixing and blending aids Lecithins decrease time

and increase efficiency of mixing of unlike ingredients

such as sugar and shortening by providing lubricity as

well as viscosity reduction at the contact surfaces of

the incompatible solids.

0014 Release agents Lecithins provide easy release from

metallic surfaces by attaching to the metal surface

during hot or cold cooking. They assist in the clean-

ing of hot surfaces where proteins or batters are

applied. They also reduce sticking between frozen

food products.

0015 Separating agents Lecithins prevent the adhesion of

products that normally stick when in contact, like

cheese slices and caramel confectionery.

0016Viscosity modifiers Lecithins reduce viscosity by

coating particles to reduce particle–matrix friction

such as in chocolates.

0017Wetting agents Lecithins provide complete wetting

of fatty or hydrophilic powders in aqueous systems.

The fatty acids are attracted to the fatty portion and

the hydrophilic portions of the molecules actively

imbibe water and control the hydration of the

powder.

Manufacture

0018The majority of phospholipids are solvent-extracted

from their source. Usually, nonpolar hydrocarbons,

like hexane, are used. Soya beans, for example, are

cleaned, cracked, and dehulled before flaking and

extraction. The hexane is removed from the solvent

micelle and the crude soya bean oil is cooled for

further refining. Figure 1 shows a flow diagram for

the degumming of crude soya bean oil and the pro-

duction of lecithin. Approximately 2–3% water is

added to the crude oil and agitated for at least

30 min. The phosphatides hydrate, swell, and are

separated by centrifugation. The wet gums with

50% moisture are dried through a thin-film drier.

The important points here are careful drying tem-

peratures and cooling of the product below 20



Lightening the color of the product can be achieved

with hydrogen peroxide in the gum stage. The hydro-

gen peroxide is removed through drying.

Modification

0019The chemistry and functionality can be altered by

simple chemical additions with acids and bases as

well as hydrogen peroxide and acetic anhydride.

These modifications increase the dispersion and hy-

dration properties of the lecithin. Enzyme modifica-

tion is also possible with lipases and phospholipases.

These changes markedly affect the functionality in

emulsification.

Composition of Lecithins

0020The composition of lecithins and their phospholipids

will vary depending on their source: vegetable,

animal, or bacterial. There are minor differences

within a class but major differences between the

sources. Vegetable lecithins are high in phosphatidyl-

choline, phosphatidylethanolamine, phosphatidyl-

inositol, and phosphatidic acid, but very low in

phosphatidylserine and contain no sphingomyelin.

Animal lecithins are high in phosphatidylcholine,

phosphatidylethanolamine, phosphatidylserine and

tbl0002 Table 2 Functionality of lecithins

Adhesion aid

Antibleed agent (as in fat bloom)

Anticorrosive

Antidusting agent

Antioxidant

Antispatter agent

Biodegradable additive

Biologically active agent

Catalyst

Color intensifier

Conditioning agent

Coupling agent

Dispersing agent, mixing aid

Emollient, softening agent

Emulsifier or surfactant

Flocculant

Grinding aid

Lubricant

Liposomal encapsulating agent

Machining aid

Modifier

Moisturizer

Nutritional supplement, vitamin source

Penetrating agent

Plasticizer

Promoter

Release agent, antisticking agent

Spreading agent

Stabilizer

Strengthening agent

Suspending agent

Synergist

Viscosity modifier

Water repellent

Wetting agent

Reproduced from Schmidt JC and Orthoefer FT (1985) In Szuhaj BF and

List GR (eds) Lecithins, p. 187. Champaign: American Oil Chemists’

Society, with permission.

4516 PHOSPHOLIPIDS/Properties and Occurrence

sphingomyelin but contain no phosphatidylinositol.

Microbes have phospholipids similar to the plant

kingdom with high levels of phosphatidylethanol-

amine and phosphatidylcholine and phosphatidyl-

serine or sphingomyelin.

Specifications of Lecithins

0021 Lecithins may be qualified in several ways, chem-

ically, physically, and functionally, but there are also

specifications used to assess quality and purity

(Table 3). These include acetone-insolubles (AI),

acid value (AV), hexane-insoluble matter (HI), perox-

ide value (PV), moisture, color, free fatty acids (FFA),

divalent metals (DVM), iodine value (IV), and phos-

phorus. Most of these analytical methods are found in

the American Oil Chemists’ Society (AOCS) Official

Methods and Recommended Practices, Section J.

Acetone-Insolubles

0022 Phospholipids are nearly insoluble in cold acetone.

This quantitative method should measure the active

ingredients in lecithin. Depending on the type of

lecithin, the range is 35–98%.

Acid Value

0023The phosphorus group in lecithins have a titratable

acidity that is measured with this volumetric method.

FFAs are also measured in this test and should not be

confused with phospholipid acidity. The AV range is

20–36 mg of potassium hydroxide per gram.

Hexane-Insolubles

0024In the processing of soya bean lecithin, particulate

matter finds its way through some processes and

gives the product a hazy appearance. The HI can

be determined by dissolving the product in hexane,

centrifuging and gravimetrically measuring the

insolubles. The HI range for soya bean lecithins is

0.05–0.3%.

Peroxide Value

0025The PV is a measure of oxidation in fats and oils. In

lecithin, however, the PV usually measures the

residual hydrogen peroxide from processing. The

range in unbleached products is 0–10 mmol kg

1

and in bleached products is 10–75 mmol kg

1

Filtered, warm, crude oil

Flowmeter

Pipeline dwell

agitator

Centrifuge

Oil

Heater

Vacuum

Condensate

Fluidity agent

Bleaching agent

Gums

Pump

Condenser

Condensate

receiver

Mixing tank

Dry lecithin

Cooler

Pump

To packaging

Agitated-film

evaporator

Vacuum drier

Cooler

Pump

To storage

Degummed

dry

soya bean oil

Flowmeter

Water

fig0001 Figure 1 Flowsheet for degumming and crude lecithin production. Reproduced from List GR (1989) In Szuhaj BF (ed.) Lecithins:

Sources, Manufacture and Uses, p. 149. Champaign: American Oil Chemists’ Society, with permission.

PHOSPHOLIPIDS/Properties and Occurrence 4517

Moisture

0026 The water content of lecithins is quite low, at

0.1–1.0%. It can be measured by oven drying but is

more accurately determined by the Karl–Fischer

method. This water content is so low that there is

no measurable water activity for microbial growth.

Moisture contents above 1% will change the viscosity

from a fluid to a plastic state. (See Water Activity:

Principles and Measurement.)

Viscosity

0027 The Brookfield viscosity at 25



C will have a range of

150–20 000 cP. Diluents and divalent metals can alter

the viscosity to usable levels.

Free Fatty Acids

0028 This method measures the true fatty acid levels in

lecithins which range from 1 to 5 mg of potassium

hydroxide per gram. Fatty acids are added as additives

to adjust the viscosity. (See Fatty Acids: Analysis.)

Iodine Value

0029 The IV is a traditional method for qualifying lecithin

sources. The more unsaturated fatty acids are found

in soya bean lecithins, which have a range of 95–110

in natural fluid lecithins, to 80–90 in deoiled lecithin.

Phosphorus

0030This wet chemical method is an indirect way of meas-

uring the phospholipid content. The typical level of

phosphorus is 2.0% in fluid lecithin and 3.0% in

deoiled products. The AOCS method Ca 12–55 has

an approximation for converting percent phosphorus

to the phosphatides in soya bean oil. The equivalent

phosphatides content is equal to percent phosphorus

30.

Uses of Lecithin

0031There are many uses of phospholipids in the food

industry. As seen from the functional properties,

there are multiple functions for lecithins. The

following is a listing of the major areas of use:

0032margarines – emulsifier, stabilizer, and antispatter;

0033confectionery and snack foods – crystallization

control, viscosity control, antisticking;

0034instant foods – wetting and dispersing agent, emul-

sifier;

0035commercial bakery products – crystallization con-

trol, emulsifier, wetting agent, release agent;

0036cheese products – emulsifier, release agent;

0037meat and poultry processing – browning agent,

phosphate dispersant;

0038dairy and imitation products – emulsifier, wetting

and dispersing agent, antispattering and release

agent;

0039packaging aid – release agent, sealant;

0040processing equipment – internal or external release

agent, lubricant.

Refer to individual foods

Applications of lecithins in foods are clearly sup-

ported by the Food Chemicals Codex, the European

E322 regulations, and they are considered as gener-

ally regarded as safe substances by the US Food and

Drugs Administration.

Storage and Handling

0041Lecithins are very stable products. They are shipped

in drums or in bulk containers. They can be stored at

ambient temperatures for up to 2 years without loss

of activity or becoming rancid or spoiling. The water

activity is so low that no microbial growth can occur.

The products may be heated to 25



C for easier appli-

cation. (See Water Activity: Effect on Food Stability.)

See also: Eggs: Structure and Composition; Emulsifiers:

Organic Emulsifiers; Fatty Acids: Properties; Analysis;

Soy (Soya) Beans: Properties and Analysis; Spices and

Flavoring (Flavouring) Crops: Tubers and Roots;

Triglycerides: Structures and Properties; Water Activity:

Principles and Measurement; Effect on Food Stability

tbl0003 Table 3 Specifications range for commercial lecithins

Analysis Typicalrange

Acetone-insolubles (%) 35–98

Acid value (mg KOH g

1

) 20–36

Hexane-insolubles (%) 0.05–0.3

Peroxide value (mmol kg

1

)

Unbleached 0–10

Bleached 10–75

Moisture (%) 0.1–1.0

Viscosity (Brookfield, 25



C) (cP) 150–20 000

Free fatty acids (mg KOH (g

1

) 1–5

Iodine value (cg I g

1

)

Natural 95–110

Oil-free 80–90

Fattyacid composition (%)

Soya bean oil Natural Oil-free

16:0

10.3 15.6 20.3

18:0

4.4 4.7 4.6

Total saturates 14.7 20.3 24.9

18:1

24.5 17.9 9.2

18:2

53.8 54.0 58.9

18:3

7.0 6.7 7.0

Total unsaturates 85.3 78.6 75.1

Unsaturated: saturated ratio 5.8:1 3.9:1 3.0:1

Reproduced from Central Soya, Chemurgy Division (1990), with

permission.

4518 PHOSPHOLIPIDS/Properties and Occurrence

Further Reading

Burner D (ed.) (1991) Official Methods and Recommended

Practices. Champaign: American Oil Chemists Society.

Central Soya, Chemurgy Division (1990) The Lecithin

Book. Fort Wayne: Central Soya.

Charalambous G and Doxastakis G (1989) Food Emulsi-

fiers: Chemistry, Technology, Functional Properties and

Applications. New York: Elsevier.

Hanin I and Pepeu G (1990) Phospholipids: Biochemical,

Pharmaceutical, and Analytical Considerations. New

York: Plenum Press.

Szuhaj BF (ed.) (1989) Lecithins: Sources, Manufacture and

Uses. Champaign: American Oil Chemists’ Society.

Szuhaj BF and List GL (eds) (1985) Lecithins. Champaign:

American Oil Chemists’ Society.

Determination

B F Szuhaj, Central Soya Company Inc., Fort Wayne,

IN, USA

This article is reproduced from Encyclopaedia of Food Science,

Introduction

0001 Phospholipids are a well-known class of lipids that

have been thoroughly analyzed over the past three

centuries. Their complete analysis was facilitated by

the great advances in separation science and qualita-

tive procedures that have occurred in the last 50

years. This article will cover the analysis of phospho-

lipids from their structure and composition, extraction

techniques, qualitative and quantitative assays, and

industrial methodology. (See Fats: Classification.)

Structure of Phospholipids

0002 There are at least a dozen compounds that fall into

the class of phospholipids. They have a basic struc-

ture of a diacylglycerol backbone with a phosphate

ester on the a or third carbon of the glycerol mol-

ecule. Usually another compound is attached that

characterizes the phospholipid. Figure 1 shows

examples of the structures of the major phospho-

lipids. These phospholipids (and their common ab-

breviations) are:

0003 phosphatidylcholine (PC, PtdCho)

0004 phosphatidylethanolamine (PE, PtdEth)

0005 phosphatidylserine (PS, PtdSer)

0006 N-acylphosphatidylethanolamine (NAPE, N-acyl-

Ptd-Eth)

0007 phosphatidylinositol (PI, PtdIns)

0008phosphatidic acid (PA, PtdA)

0009phosphatidylglycerol (PG, PtdGro)

0010plasmologen (PM)

0011diphosphatidylglycerol (DPG, diPtdGro)

0012lysophosphatidylcholine (LPC, lysoPtdCho)

0013lysophosphatidylethanolamine (LPE, lysoPtdEth)

The proper nomenclature for phospholipids has been

defined by the 1976 revised recommendations of the

International Union of Pure and Applied Chemistry

(IUPAC) and the International Union of Biochemis-

try (IUB) Committee on Biochemical Nomenclature.

For example, the term ‘lecithin’ is permitted for

phosphati-dylcholine but the systematic name is

1,2-diacyl-sn -glycero-3-phosphorylcholine. The gen-

eric name of 3-sn-phosphatidylcholine could be used.

The abbreviation PtdCho is also allowed. This article

will use the common names listed above since

the literature has thousands of references with this

terminology.

Composition of Phospholipids

0014The composition of phospholipids depends on the

source of the phospholipids. Those from animal,

plant, and microbial sources will have different com-

positions, depending on the nature of the tissue from

which the lipids are extracted – for example, brain,

liver, or blood. In plants, it will vary on whether they

are from soya beans, corn, cotton, rapeseed, or sun-

flower. In microbial sources it depends upon the

organism.

0015The phospholipid classes are similar within a

species, but differ primarily in fatty acid acyl compos-

ition around the 1 and 2 positions on the glycerol

backbone.

Fatty Acids

0016The fatty acid chain length is commonly from C

, with different degrees of unsaturation from one

to six double bonds, which may be at different loca-

tions on the acyl group. However, there is a pattern

that is relevant to the present discussion. (See Fatty

Acids: Properties.)

0017In animals the primary fatty acids range from C

12:0

(lauric acid) to C

24:0

(tetracosanoic acid). Again,

depending on the species and tissue extracted, the

fatty acids can be variable.

0018In the plant kingdom, the primary diacyl groups on

the phospholipids will range from C

12:0

to C

18:3

, i.e.,

lauric to linolenic acid. There are usually no C

fatty

acids and higher, as in the animal kingdom. The degree

of unsaturation depends on the origin of the crop, i.e.,

from temperate or tropical regions. Also, the climate

within the zone can make a seasonal difference.

PHOSPHOLIPIDS/Determination 4519

0019 Microbial phospholipid acyl groups are more simi-

lar to those found in the plant kingdom than they are

to those from the animal kingdom. The predominant

acyl fatty acids range from C

16:0

to C

18:3

. There are

more odd-numbered carbon fatty acids in micro-

organisms than elsewhere.

0020 A further factor that makes phospholipid compos-

ition more complex is the ability to have different

fatty acids on the 1 and 2 positions of the molecule.

Most research has shown that polyunsaturated fatty

acids are usually in the 2 position.

Extraction Techniques

0021 One of the most important factors in phospholipid

analysis is the initial extraction procedure. If the

analysis is on a finished commercial lecithin there is

no problem, but if the analysis is from tissue samples

or food samples the extraction technique will be crit-

ical in obtaining meaningful results.

Tissue Samples

0022For many years the Folch extraction of tissue hom-

ogenates with chloroform/methanol 2:1 (v/v) has

been the method of choice by most researchers.

Some found that the use of chloroform/methanol 1:1

(v/v) was preferable and some have used a biphasic

system of butanol/methanol with dilute hydrochloric

acid. Some have used hexane/2-propanol 3:2 (v/v).

Which solvent system used depends largely on the

required accuracy, but in most cases chloroform/

methanol 2:1 (v/v) is the best solvent to try initially.

Phosphatidylcholine

Phosphatidylserine

Phosphatidylinositol

Phosphatidylglycerol

Diphosphatidylglycerol

N-Acylphosphatidylethanolamine

Phosphatidic acid (PA)

Plasmalogen

X = Choline or ethanolamine

Phosphatidylethanolamine

−

N (CH

)

OCOR

COCO

COPO

−

CHNH

OCOR

COCO

COPO

−

COO

−

CHNH

OCOR

COCO

CO O PO

−

OCOR

COCO

COPO

−

HCOH

COH

OCOR

COCO

COPO

−

HCOH

OCOR

−

COPO

C O CO R

HC O CO R

COCH

COPO

−

OCOR

CHR

COCO

COPO

−

OCOR

COCO

COPO

−

CO R

OCOR

fig0001 Figure 1 Structures of the major phospholipids. Reproduced from Scholfield CR (1985) In: Szuhaj BF and List GR (eds)Lecithins, p.3.

Champaign: American Oil Chemists’ Society, with permission.

4520 PHOSPHOLIPIDS/Determination

Food Samples

0023 Since food samples may have lecithin or phospholipid

added rather than being incorporated into the tissues

of the food matrix, the dried sample can be ground

and extracted with petroleum ether. If the lipids are

bound in the product through processing, chloro-

form/methanol 2:1 (v/v) can be used. Because of

environmental considerations di- or trichloroethane

should be used in place of chloroform.

0024 Drying of high-moisture products is required, but

not by oven drying or air drying, as oxidation of the

fatty acids can occur. Freeze drying of the product is

preferred.

Qualitative Analysis

0025 There are several ways to detect the presence of phos-

pholipids. Traditional instrumental methods using

ultraviolet and infrared are not used as often since

thin-layer chromatography (TLC) can give a qualita-

tive and semiquantitative result in one assay. Also, the

use of conformational chemical sprays on the TLC

plates can further identify the products.

Thin-Layer Chromatography

0026 There are several types of silica gel plates available for

TLC. Silica gel G and H are the most useful. Phos-

pholipids may be separated on a 20 20 cm plate in

one or two directions. A polar solvent and a nonpolar

solvent system are used. The polar system is chloro-

form/methanol/water (65:25:4, v/v/v) and the nonpo-

lar solvent is petroleum ether/diethyl ether/acetic acid

(90:10:1, v/v/v). See the American Oil Chemists’

Society (AOCS) recommended practice Ja 7-86

for alternative methodology. (See Chromatography:

Thin-layer Chromatography.)

0027 These TLC plates are air- or oven-dried after separ-

ation of 20–50 mg of sample and are sprayed with

10% sulfuric acid and heated to char the phospho-

lipids. Alternatively, they may be sprayed with a

phosphorus spray containing molybdenum blue.

Phospholipids stain a deep blue on heating the TLC

plate. An example is shown in Figure 2.

0028 Nondestructive visualization techniques can be

used if the phospholipids are to be determined or the

fatty acid composition is to be run. Ultraviolet light

and 2

-dichlorofluorescein easily detects lipids on

TLC. On prep plates, the bands are scraped off and

extracted with chloroform/methanol (1:1, v/v) and

the fatty acids converted to methyl esters using

boron trifluoride and then determined using gas–

liquid chromatography (GLC).

Quantitative Analysis of Phospholipids

Column Chromatography

0029Column chromatography precedes TLC in the separ-

ation of phospholipids. The techniques are slow and

require good skill with column preparation, flow

rates, and solvent removal. Commercial lecithins

can be separated by dissolving the crude mixture in

petroleum ether and passing it through a deactivated

silica gel column with petroleum ether. The phospho-

lipids are adsorbed and do not pass through the

column, whilst triglycerides and sterol esters are

eluted. The phospholipids are subsequently quanti-

fied by TLC and wet phosphorus analysis.

High-Performance Liquid Chromatography (HPLC)

0030Newer technologies have found that HPLC can sep-

arate and quantify phospholipids more quickly and

accurately. Separation is carried out on several types

of columns, including silica gel and an amino group

bonded to the silica surface (mBondapak-NH

). The

columns are eluted with chloroform/methanol gradi-

ents, acetonitrile/methanol/85% phosphoric acid, or

acetonitrile/methanol/ water. The eluent is measured

at 205 nm or detected with flame ionization. Figure 3

Acylated

lecithin

Ori

NAPE

LysoPE

LysoPC

fig0002Figure 2 Thin-layer chromatogram of soya bean lecithin in two

dimensions: triglycerides (TG), sterol glucosides (SG), phospha-

tidic acid (PA), N-acylphosphatidylethanolamine (NAPE), phos-

phatidylethanolamine (PE), lysophosphatidylethanolamine (Lyso

PE), phosphatidylcholine (PC), phosphatidylinositol (PI), lyso-

phosphatidylcholine (LysoPC). Silica gel plate; first dimension

chloroform/methanol/acetic acid/water, 85:15:15:3 (v/v/v/v);

second dimension chloroform/acetone/methanol/acetic acid/

water (10:4:2:2:1) (v/v/v/v/v). Courtesy of J. Yaste, Central Soya,

Food Research, Fort Wayne, Indiana, USA.

PHOSPHOLIPIDS/Determination 4521

shows an HPLC separation of commercial lecithin,

using ultraviolet detection. The mass detector,

an evaporative analyzer, has also been successfully

used for the HPLC determination of phospholipids.

(See Chromatography: High-performance Liquid

Chromatography.)

Densitometry

0031 Densitometric scanning has been used as an indirect

method for determining phospholipid content on TLC

plates. While the method has some promise, a problem

is the quanitative charring of the phospholipid spots.

Each phospholipid has a different charring density and

this depends on fatty acid composition. Only with

proper standards can this method be useful.

Thincography

0032 Thin-rod TLC combines TLC with quantification by

flame ionization detection. Rods are used rather than

plates but controversy still exists over the suitability

of the technique for routine lipid analyses.

Phosphorus Analysis on Phospholipids

0033 Phosphorus analysis is an indirect method for

the quantification of phospholipids because the

qualitative composition of the sample must be

known, if accurate values are to be obtained. With

pure phosphatides this will work well, but most sep-

aration techniques give mixed phospholipids. The

preferred method for phosphorus in lecithins is the

AOCS method Ja 5-55. This determines the total

phosphorus content of the sample. For commercial

lecithins a multiple factor of 30 is used to convert

total phosphorus values to acetone-insoluble value.

0034There are various methods to determine phos-

phorus through molybdenum blue and molybdova-

nadophosphate yellow. To improve reproducibility

many factors need to be evaluated. This includes the

digestion method, chromogenesis, and sensitivity.

The AOCS method is the most straightforward and

should be used especially in the food area.

Industrial Methods of Analysis

0035Phospholipids are characterized by a different set of

assays than the determination of compounds used for

academic or biochemical use. Most commercially

available phospholipids come from soya beans and

from eggs, and the methods outlined below can be

used to qualify or categorize the products.

Acetone-Insolubles (AI)

0036This method determines the content of phospho-

lipids in commercial lecithins. The method employs

AOCS method Ja 4-46. The AI is an approximation of

the active ingredients in formulations. Cold lecithin-

saturated acetone must be used in this test.

Acid Value (AV)

0037This method determines the phosphatide and fatty

acid content of commercial phospholipids. The

method utilized is AOCS method Ja 6-55. Phospha-

tide acidity is often confused with fatty acid addition

to commercial lecithin. It is a combination of organic

acids and phosphoric acid.

Peroxide Value (PV)

0038This is a measurement of oxidative state of commer-

cial phospholipids. It measures the milliequivalents of

peroxide per kilogram of sample which oxidize po-

tassium iodide. It also measures the residual peroxide

used in process stabilization and bleaching. AOCS

method Ja 8-87 is used.

Free Fatty Acids (FFA)

0039This method utilizes AOCS method Ca 5a-40. When

run on the acetone-soluble portion of the AI method,

it gives the added fatty acids.

PC (RT 23.68)

PA ( RT 6.65)

PI ( RT 8.75)

PE (RT 4.35)

Deoiled lecithin 83.2 mg 50 ml

−1

hexane

fig0003 Figure 3 High-performance liquid chromatography of deoiled

soya bean lecithin. PC, phosphatidylcholine; Pl, phosphatidylino-

sitol; PA, phosphatidic acid; PE, phosphatidylethanolamine.

Column, m Porasil 10 m 3.98 300 mm; mobile phase, hexane/2-

propanol/acetate (8:8:1, v/v/v), buffer pH 4.2; detection, ultraviolet

(206 nm); injection, 10 ml; flow rate, 1 ml min

1

. Retenton time (RT)

in minutes. Courtesy of P. Balazs, Central Soya, Food Research,

Fort Wayne, Indiana, USA.

4522 PHOSPHOLIPIDS/Determination

Phosphorus Content

0040 The determination of total phosphorus is an indirect

method for quantifying phospholipids. This method

(AOCS method Ja 5-55) quantifies phospholipids

through a molybdate reaction to a chromophore

quantitated by phenolphthalein titration.

Gas Chromatography (GC)

0041 This method is commonly used to measure the fatty

acid composition and does not quantify the phospho-

lipids themselves (AOCS method Ce 1-62).

High-performance Liquid Chromatography

0042 This is gradually replacing the older techniques for

qualifying and quantifying particular phospholipids

in commercial mixtures. A uniform technique is being

addressed by the AOCS.

0043 Phospholipid analyses have come a long way since

their study by Theuticum, c. 1800. Each analyst must

choose the best method depending on constraints of

accuracy and time.

See also: Chromatography: Thin-layer Chromatography;

High-performance Liquid Chromatography; Fatty Acids:

Properties; Fats: Classification