Lipids form a broad category of biomolecules which constitute an essential part of living organisms in addition to carbohydrates and proteins. In this section, we will introduce lipid related substances such as fatty acids and their derivatives. The biosynthesis of fatty acids involves the condensation of malonyl-CoA (or methylmalonyl CoA) with acyl CoA as a primer.¹⁾ Carboxylic acids with chains of 4 or more carbons are referred to as fatty acids while those with 10 or more carbons are called higher fatty acids.²⁾ Lipids and related substances can be classified as illustrated in this section.³⁾

Steroids or terpenes which are occasionally considered as lipid related substances will be explained in other sections.

■Functional Roles
Fatty acids exist in living organisms mainly as esters of glycerol and triacylglycerols which occur as a major form of energy storage in adipose tissue. Triacylglycerols are found in living organisms as mixtures of acyl groups with different number of carbons and are difficult in most cases to isolate as a single substance. Moreover, fatty acids also exist in living organisms in the form of cholesteryl esters which constitute an essential component of cell membranes where it is required to establish proper membrane permeability and fluidity.
Free fatty acids are known to suppress cell-growth at an order of 0.1mM and above, therefore considerable attention should be paid during their administration to cells.⁴⁾

■Derivatives
Fatty acid methyl esters are the most widely used fatty acid derivatives in analytical chemistry due to their ease of handling in organic solvents as compared to the highly polar free fatty acids. Moreover their analysis by TLC, gas chromatography (GC) and liquid chromatography (LC) can be improvised by suppressing tailing.
Although fatty acid ethyl esters are rarely used for analysis as compared to the methyl esters, they offer an advantage since their method of preparation from fatty acids involves the use of the less toxic ethanol instead of methanol. For instance, eicosapentaenoic acid which is used as a hyperlipemia medicine is being supplied as ethyl ester. Ethyl esters of lower to middle-chain fatty acids can be also employed in the fragrance industry.
Sodium salts of fatty acids can be obtained as saponification products of lipids and are widely used in daily life as an ingredient of soap owing to their amphiphilicity and surfactant properties.

■Analysis
GC is the most frequently used technique for the analysis of fatty acids. However, their direct analysis appears to be difficult owing to their low volatility in electron impact (EI) ionization mass spectrometry under normal condition. They can however be easily detected by GC-MS method upon derivatization to their methyl esters.
The several methylation methods include the treatment of the free fatty acids with boron trifluoride-ether complex in methanol or with trimethylsilyldiazomethane, etc.
The GC on-column method is a simple method that uses a methanolic solution of trimethyl sulfonium hydroxide or 3-(trifluoromethyl)phenyltrimethylsilyl ammonium hydroxides to analyze the fatty acid component of lipids such as glycerolipids. Please refer to "GC Derivatizing Reagents" for protocols.

■Solubility
In general, the solubility of fatty acids in water decreases as the carbon number increases.
Fatty acid esters and glycerolipids are insoluble in water but soluble in ethanol, chloroform and diethyl ether. They can be added to the buffer solution as a dimethyl sulfoxide solution to examine their activity in living organisms. Please take caution that the solution becomes suspended as the concentration level of the dissolved substance increases. It is recommended to define the optimal concentration level and volume of addition in advance. Sodium salts of fatty acid are more water-soluble than the free fatty acids. Long-chain fatty acid salts tend to form micelles.

■Storage Precautions
Unsaturated fatty acids like oleic acid are known to undergo aerial oxidation to produce peroxides. Opened bottles of unsaturated fatty acids and their derivatives should be stored in the refrigerator or frozen with inert gas such as nitrogen or argon. Moreover the tendency to oxidation increases as the degree of unsaturation in the fatty acids increases.

Saturated Fatty Acids

Unsaturated Fatty Acids

Fatty Acid Methyl Esters

Fatty Acid Ethyl Esters

Fatty Acid Sodium Salts

Saturated Higher Alcohols

Unsaturated Higher Alcohols

Glycerolipids

Fatty Acid Cholesteryl Esters

Waxes

Lipid Extracts

Glycerophospholipids

Sphingolipids

Saturated Fatty Acids

E0006	B1248	B1747
D0963	B0754	C0829
D0017	H0010	H0971
H0019	H0030	H0105
L0011	T0076	M0595
M0476	N0662	N0283
N0288	M1342	O0027
P0002	P0882	P0035
S0163	T0402	T0412
U0004	V0003

Unsaturated Fatty Acids

E0441	A0781	C0416
D0098	D1186	D2226
O0010	D0965	H0426
H0427	H0072	H0428
H0077	H0383	L0053
L0124	L0050	L0152
N0312	N0426	O0004
O0070	O0011	O0180
P0345	P1072	O0009
S0053	T1642	T0759
U0032

Fatty Acid Methyl Esters

A0650	A0900	B1241
B1748	B0763	C0419
D0023	D1017	H0566
H0032	P1958	H0111
H0704	H0705	H0835
L0015	L0112	L0078
L0051	M0482	N0460
N0290	O0226	O0033
M2014	O0055	O0246
K0037	K0035	M1863
P0006	P0869	P1210
R0029	S0056	S0080
T0812	T0960	U0050
V0005

Fatty Acid Ethyl Esters

A0649	D2964	E0442
E0853	A0899	B0759
C0418	D0022	D1931
D2767	D2195	D1014
E0345	H0526	H0031
H0501	H0108	E0787
H0229	H0230	H0975
H1029	E0644	L0013
E0802	L0120	L0111
L0055	L0135	E0801
E0803	M0479	N0459
N0289	O0030	O0286
O0054	O0143	O0216
K0030	K0031	P0003
P0868	R0049	S0079
T0959	U0049	V0004

Fatty Acid Sodium Salts

S0519	D0024	S0862
E0009	C1187	H0112
L0016	L0056	M0483
N0291	O0034	O0057
P0007	R0030	S0058
S0081

Saturated Higher Alcohols

D0964	D0978	E0004
H0011	H0018	H0342
H0071	N0284	O0199
O0006	P0036	T0593
T0084	T1049	T0403
T0803	U0005

Unsaturated Higher Alcohols

O0225	D2174	D2294
D1936	O0228	O0058
T1502

Glycerolipids

G0079	M1072	M1071
M1075	M1076	G0081
M1073	G0082	G0083
G0085	T1389	T1391
T0364	T0413	T0441
T0365	T1392	T1390
G0087	T1388	G0088
G0089	G0091	G0213
G0212

Fatty Acid Cholesteryl Esters

C0319	C0668	C0618
C0672	C0673	C0620
C0321	C0675	C0334
C0559	C0322	N0347
C0676	C0323	C0677

Waxes

S0078

P1077

Lipid Extracts

Glycerophospholipids

D3924

D3946

D3925

Sphingolipids

P1765