Gas chromatographic retention data of wax esters

Journal of Chromatography A, 1128 (2006) 208–219

Gas chromatographic retention data of wax esters Karel Str´ansk´y, Marie Zarev´ucka, Irena Valterov´a ∗ , Zdenˇek Wimmer ∗ Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Department of Natural Products, Flemingovo n´am. 2, CZ-166 10 Prague 6, Czech Republic Received 6 February 2006; received in revised form 8 June 2006; accepted 9 June 2006 Available online 3 July 2006

Abstract Higher wax esters within the range of C24 to C44 (205 standards) were analyzed by means of gas chromatography and Kov´ats indexes (I) and reduced Kov´ats indexes (RKI) were calculated. The dependences of these retention data on number of carbon atoms and on number and position of double bonds in acid and in alcohol moieties of esters were plotted. © 2006 Elsevier B.V. All rights reserved. Keywords: Lipids; Higher esters; Wax esters; Fatty acids; Fatty alcohols; Gas chromatography; Kov´ats index; Reduced Kov´ats index

1. Introduction Higher esters, also called wax esters, are present not only in lipids of almost all plants, animals and insects but also in fossil materials [1–5]. Even if a number of them have already been isolated and identified at the beginning of the previous century, more detailed analyses of this group of lipids could only be done with broader availability of chromatographic techniques, namely column chromatography and thin-layer chromatography in combination with gas chromatography. The compounds studied may then be identified by means of mass spectrometry techniques. Higher esters of plants are located in wax cuticular layer [1–5]. A number of insect families also bear special glands producing waxes (beeswax, ghedda, shellac or Chinese waxes) and together with selected plant waxes (bayberry, candelilla, carnauba, coffee berry, cork, cotton, esparto, flax, hemp, Japan, lemon, ouricury, retamo or sugar cane waxes, jojoba and rice bran oils) have become important industrial raw materials. Higher esters are also components of animal sebum [1,2,5] and/or of secretion of special glands [6]. All birds with a few exceptions also produce a secretion by the uropygial (preen) gland; the basis of this secretion also consists in esters [2,7]. A sperm oil (spermaceti) has been obtained from bodies of sev∗

Corresponding authors. Tel.: +420 220 183 281; fax: +420 220 183 582. E-mail addresses: [email protected] (I. Valterov´a), [email protected] (Z. Wimmer). 0021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2006.06.035

eral sea mammal species [1,2,5,8,9], and esters from small sea animals and plants (plankton) are essential substances in food chain [2,10–13]. Fossil waxes (peat wax and montan wax) are of plant origin [1,5,14]. Higher esters isolated from extracts are relatively complex mixtures of compounds, which are quite similar one to another. In our previous paper [15] we published that the total number of acids from C14 till C25 (saturated and unsaturated straight chain isomers and homologues with the Z-configuration of double bonds, methylene interrupted) is represented by a number of 756 compounds. If the same type of variability occurred in the alcohol moiety, the total number of esters would theoretically limit to several tens thousands individual compounds. No study has yet been done, which would investigate systematically either gas chromatographic properties of such individuals or their mass spectra. We have focused attention on obtaining gas chromatographic retention data of the individual higher esters during the first part of our project, while the investigation of their mass spectra will be a subject of another paper in a future. 2. Experimental 2.1. Reference compounds A majority of the reference samples was purchased from the Nu-Chek-Prep, Inc., Elysian, MN, USA. Methyl, ethyl, propyl, butyl, hexyl, octyl, and decyl esters of several acids were syn-

K. Str´ansk´y et al. / J. Chromatogr. A 1128 (2006) 208–219

thesized in our department in past years as mixtures of several individuals in each group. 2.2. Synthesis of acetates 1-Alkanols with even number of carbon atoms (C22 , C24, C26 , C28 and C30 ; 1 mg of each 1-alkanol) and 4-dimethylaminopyridine (Aldrich-Chemie, Steinheim, Germany) (5 mg) were dissolved in CHCl3 (3.5 ml), and after addition of acetyl chloride (100 ␮l) the mixture was stirred at 30 ◦ C. The reaction course was monitored by TLC. After completing of the reaction (2 h), the reaction mixture was transferred into a separation funnel. Water (3 ml) was added, and the organic layer was extracted with CHCl3 (3 × 2 ml). Collected extracts were washed with water (3 × 2 ml), and dried over MgSO4 . The mixtures of acetates were obtained by removing of the solvent under reduced pressure. Acetates derived from a mixture of odd 1-alkanols (C23 , C25 , C27 , C29 and C31 ) were prepared accordingly. 2.3. Thin-layer chromatography (TLC) 2.3.1. Analytical TLC Analytical TLC was carried out on glass plates (76 mm × 36 mm) coated with silica gel Adsorbosil-Plus (Applied Science Division, State College, PA, USA) with gypsum (12%, w/w), layer thickness 0.2 mm. Detection of the spots was achieved by spraying the TLC plates with concentrated H2 SO4 and by subsequent heating. 2.3.2. Preparative TLC Preparative TLC (PTLC) was carried out on glass plates (76 mm × 60 mm) coated with the same silica gel type as above. Reaction mixtures resulting from the syntheses of methyl, ethyl, propyl, butyl, hexyl, octyl, and decyl esters (3 mg of each mixture) were applied separately on the TLC plates and eluted by a light petroleum (b.p. 40–60 ◦ C)/diethyl ether mixture (94:6, v/v, both solvents freshly distilled). The TLC plates were sprayed with Rhodamine 6G (Merck AG, Darmstadt, Germany; 0.05% solution in ethanol) and visualized in UV (254 nm). The chromatographic bands, containing esters, were scraped off and transferred into small columns (8 mm i.d.) filled with silica gel (0.3 g, particle size 25–50 ␮m), eluted with dry diethyl ether (15 ml, freshly distilled) and evaporated to dryness. 2.4. Gas chromatography (GC) GC was performed with a HP 5890A gas chromatograph (Hewlett-Packard Co., Avondale, PA, USA) equipped with a flame ionization detector and split/splitless injector. The injector was used in a split mode with a SGE 4 mm I.D. FocusLiner filled with quartz wool (SGE, Ringwood, Australia), both silanized by dimethyldichlorosilane in toluene (20%, v/v, 25 ◦ C, 30 min). Manual injection was performed with a 10 ␮l Hamilton syringe. Higher esters and n-alkanes were co-injected. Chromatographic conditions: injector and detector temperatures 260 ◦ C, oven temperature 240 ◦ C (0 min), than a rate 1 ◦ C min−1 to 340 ◦ C, carrier gas hydrogen, 130 kPa, u¯ 60 cm/s at 240 ◦ C (Hydrogen

209

Generator Model 7525, Packard Instrument Co., Inc., Downers Grove, IL, USA), cleaning of carrier by moisture trap (activated ˚ hydrocarbon and oxygen traps (Supelco, molecular sieve 5 A), Bellefonte, PA, USA) and indicating oxygen trap (J&W Scientific, Folsom, CA, USA), split ratio 29:1. Nitrogen was used as make-up gas (28 ml/min). A DB-1 ms fused silica column (30 m × 0.252 mm, film thickness 0.25 ␮m, J&W Scientific) was used. Data were collected with a HP 3393A integrator (HewlettPackard). 2.5. Gas chromatography–mass spectrometry (GC–MS) Esters synthesized in our laboratory were analyzed using a gas chromatograph (Focus, Thermo, Finnigan Italia, Milan, Italy) with a split/splitless injector and a mass detector (Fisons MD 800, VG Masslab, Manchester, UK), working in electron ionization mode. Chromatographic conditions: injector temperature 220 ◦ C, interface temperature 200 ◦ C, oven temperature 160 ◦ C (0 min), than a rate 5 ◦ C/min to 320 ◦ C (20 min), carrier gas helium at constant flow rate 1 ml/min, split ratio 10:1. A DB-5 ms fused silica column (30 m × 0.25 mm, film thickness 0.25 ␮m J&W Scientific) was used. 2.6. Calculation of the Kov´ats indexes I The program for data processing for the calculating of the Kov´ats indexes (I) was written in GW-BASIC (Microsoft, Silicon Valley, Mountain View, CA, USA) [15]. The number of experimental points (n) was at least 5 with several exceptions. Outliers were automatically excluded on the basis of the Dixon test (significance level p = 0.01), when values of I were higher than calculating mean values. From the total 1155 calculated I values only 26 (2.25%) values were outliers. 2.7. Abbreviations and nomenclature of wax esters For simple and unambiguous naming of the esters in the Tables and the Figures, we have used abbreviated nomenclature for both parts of the molecule, which is commonly used in biochemistry for both, saturated and unsaturated (methylene interrupted) fatty acids. Thus, for instance, abbreviation 16:0–18:0 is used for hexadecyl octadecanoate (palmityl stearate), 16:0–18:1n-9 is used for hexadecyl (9Z)octadecenoate (palmityl oleate), 24:0–2:0 is used for tetracosanoyl ethanoate (lignoceryl acetate), 2:0–18:0 is used for ethyl octadecanoate (ethyl stearate), or 1:0–18:0 is used for methyl octadecanoate (methyl stearate). 3. Results and discussion In the past, a simple procedure was commonly used during the analyses of fatty acids and other compounds present in the lipids. The lipid part or the whole extract containing lipids was subjected to saponification. After group separation of the obtained mixture of compounds, the studied homologues were identified either by GC or by GC–MS. By this way, only the basic building blocks, i.e., hydrocarbons, acids, alcohols, sterols,

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Table 1 Higher esters and their retention data

Table 1 (Continued ) Compound

Number of carbon atoms (NCA)

Kov´ats index (I)

STD

n

Reduced Kov´ats index (RKI)

14:1n-5-20:0 14:1n-5-22:0

34 36

3541.84 3743.34

0.166 0.259

5 5

141.84 143.34

16:1n-7-12:0 16:1n-7-14:0 16:1n-7-16:0 16:1n-7-18:0 16:1n-7-20:0 16:1n-7-22:0

28 30 32 34 36 38

2932.01 3129.99 3328.91 3528.92 3730.26 3931.84

0.028 0.066 0.149 0.084 0.143 0.289

4 4 5 5 5 5

132.01 129.99 128.91 128.92 130.26 131.84

18:1n-9-12:0 18:1n-9-14:0 18:1n-9-16:0 18:1n-9-18:0 18:1n-9-20:0 18:1n-9-22:0

30 32 34 36 38 40

3123.53 3321.79 3520.87 3720.72 3922.41 4124.32

0.075 0.067 0.123 0.181 0.116 0.184

5 5 5 5 5 5

123.53 121.79 120.87 120.72 122.41 124.32

12:0-18:2n-6 14:0-18:2n-6 16:0-18:2n-6 18:0-18:2n-6 20:0-18:2n-6 22:0-18:2n-6

30 32 34 36 38 40

3116.66 3315.74 3515.52 3716.27 3918.43 4120.82

0.155 0.106 0.114 0.046 0.134 0.367

5 5 5 5 5 5

116.66 115.74 115.52 116.27 118.43 120.82

18:2n-6-12:0 18:2n-6-14:0 18:2n-6-16:0 18:2n-6-18:0 18:2n-6-20:0 18:2n-6-22:0

30 32 34 36 38 40

3118.21 3316.90 3516.49 3717.06 3919.22 4121.72

0.270 0.261 0.140 0.194 0.273 0.218

5 5 5 5 5 5

118.21 116.90 116.49 117.06 119.22 121.72

14:1n-5-14:1n-5 16:1n-7-14:1n-5 18:1n-9-14:1n-5

28 30 32

2932.37 3121.54 3213.80

0.108 0.124 0.063

6 6 5

132.37 121.54 113.80

14:1n-5-16:1n-7 16:1n-7-16:1n-7 18:1n-9-16:1n-7

30 32 34

3121.35 3310.31 3503.04

0.145 0.112 0.157

5 5 5

121.35 110.31 103.04

14:1n-5-18:1n-9 16:1n-7-18:1n-9 18:1n-9-18:1n-9

32 34 36

3314.12 3502.79 3695.89

0.185 0.125 0.327

5 5 5

114.12 102.79 95.89

142.52 140.65 140.55 142.10 143.66

12:0-18:3n-3 14:0-18:3n-3 16:0-18:3n-3 18:0-18:3n-3 20:0-18:3n-3 22:0-18:3n-3

30 32 34 36 38 40

3123.04 3322.43 3522.64 3723.93 3926.60 4129.59

0.074 0.149 0.082 0.106 0.179 0.328

6 6 6 6 5 5

123.04 122.43 122.64 123.93 126.60 129.59

5 5 5 5 5 4

131.17 129.40 128.75 128.96 130.31 132.05

18:3n-3-12:0 18:3n-3-14:0 18:3n-3-16:0 18:3n-3-18:0 18:3n-3-20:0 18:3n-3-22:0

30 32 34 36 38 40

3124.42 3323.48 3523.60 3724.49 3927.21 4130.13

0.166 0.064 0.050 0.051 0.198 0.132

5 5 4 4 5 5

124.42 123.48 123.60 124.49 127.21 130.13

0.161 0.103 0.248 0.127 0.233 0.079

5 5 5 5 5 4

122.55 121.09 120.42 120.44 122.10 124.07

14:1n-5-18:2n-6 16:1n-7-18:2n-6 18:1n-9-18:2n-6

32 34 36

3307.85 3497.40 3690.81

0.176 0.151 0.191

5 5 5

107.85 97.40 90.81

18:2n-6-14:1n-5 18:2n-6-16:1n-7 18:2n-6-18:1n-9

32 34 36

3308.64 3497.96 3691.23

0.1447 0.128 0.090

5 5 4

108.64 97.96 91.23

0.172 0.111 0.136 0.119

5 5 5 5

143.36 141.70 140.74 140.46

12:0-20:4n-6 14:0-20:4n-6 16:0-20:4n-6 18:0-20:4n-6

32 34 36 38

3259.96 3459.60 3660.65 3863.07

0.242 0.182 0.271 0.177

7 7 7 7

59.96 59.60 60.65 63.07

Compound

Number of carbon atoms (NCA)

Kov´ats index (I)

STD

n

Reduced Kov´ats index (RKI)

12:0-12:0 14:0-12:0 16:0-12:0 18:0-12:0 20:0-12:0 22:0-12:0

24 26 28 30 32 34

2554.05 2752.66 2951.62 3150.85 3351.81 3552.51

0.030 0.033 0.170 0.212 0.219 0.122

6 6 6 6 6 6

154.05 152.66 151.62 150.85 151.81 152.51

12:0-14:0 14:0-14:0 16:0-14:0 18:0-14:0 20:0-14:0 22:0-14:0

26 28 30 32 34 36

2751.90 2950.07 3148.72 3348.04 3548.72 3749.34

0.070 0.172 0.158 0.034 0.183 0.247

5 5 5 4 5 5

151.90 150.07 148.72 148.04 148.72 149.74

12:0-16:0 14:0-16:0 16:0-16:0 18:0-16:0 20:0-16:0 22:0-16:0

28 30 32 34 36 38

2950.68 3148.37 3347.13 3546.52 3747.02 3947.81

0.034 0.126 0.112 0.319 0.193 0.142

4 5 5 5 5 5

150.68 148.37 147.13 146.52 147.02 147.81

12:0-18:0 14:0-18:0 16:0-18:0 18:0-18:0 20:0-18:0 22:0-18:0

30 32 34 36 38 40

3150.19 3347.94 3546.45 3745.68 3946.15 4146.65

0.103 0.033 0.091 0.017 0.056 0.269

5 4 5 4 4 5

150.19 147.94 146.45 145.68 146.15 146.65

12:0-20:0 14:0-20:0 16:0-20:0 18:0-20:0 20:0-20:0 22:0-20:0

32 34 36 38 40 42

3350.36 3547.92 3746.49 3945.99 4146.16 4346.67

0.192 0.131 0.064 0.178 0.118 0.151

6 6 6 6 6 6

150.36 147.92 146.49 145.99 146.16 146.67

12:0-22:0 14:0-22:0 16:0-22:0 18:0-22:0 20:0-22:0 22:0-22:0

34 36 38 40 42 44

3550.68 3748.29 3946.89 4146.31 4346.38 4547.32

0.252 0.109 0.046 0.128 0.200 0.080

6 6 5 6 6 5

150.68 148.29 146.89 146.31 146.38 147.32

12:0-14:1n-5 16:0-14:1n-5 18:0-14:1n-5 20:0-14:1n-5 22:0-14:1n-5

26 30 32 34 36

2742.52 3140.65 3340.55 3542.10 3743.66

0.270 0.017 0.145 0.179 0.098

5 4 5 5 5

12:0-16:1n-7 14:0-16:1n-7 16:0-16:1n-7 18:0-16:1n-7 20:0-16:1n-7 22:0-16:1n-7

28 30 32 34 36 38

2931.17 3129.40 3328.75 3528.96 3730.31 3932.05

0.109 0.170 0.139 0.067 0.086 0.076

12:0-18:1n-9 14:0-18:1n-9 16:0-18:1n-9 18:0-18:1n-9 20:0-18:1n-9 22:0-18:1n-9

30 32 34 36 38 40

3122.55 3321.09 3520.42 3720.44 3922.10 4124.07

14:1n-5-12:0 14:1n-5-14:0 14:1n-5-16:0 14:1n-5-18:0

26 28 30 32

2743.36 2941.70 3140.74 3340.46

K. Str´ansk´y et al. / J. Chromatogr. A 1128 (2006) 208–219

211

Table 1 (Continued )

Table 1 (Continued ) Compound

Number of carbon atoms (NCA)

Kov´ats index (I)

STD

n

Reduced Kov´ats index (RKI)

20:0-20:4n-6 22:0-20:4n-6

40 42

4066.48 4270.34

0.295 0.369

7 6

66.48 70.34

14:1n-5-18:3n-3 16:1n-7-18:3n-3 18:1n-9-18:3n-3

32 34 36

3314.99 3504.89 3698.65

0.022 0.146 0.116

4 5 5

114.99 104.89 98.65

18:3n-3-14:1n-5 18:3n-3-16:1n-7 18:3n-3-18:1n-9

32 34 36

3315.81 3505.81 3699.58

0.182 0.153 0.127

5 5 5

115.81 105.81 99.58

18:2n-6-18:2n-6

36

3685.73

0.117

5

85.73

14:1n-5-20:4n-6 16:1n-7-20:4n-6 18:1n-9-20:4n-6

34 36 38

3452.29 3642.73 3837.23

0.050 0.253 0.245

4 5 5

52.29 42.73 37.33

18:2n-6-18:3n-3

36

3693.85

0.298

5

93.85

18:3n-3-18:2n-6

36

3694.05

0.085

4

94.05

18:2n-6-20:4n-6

38

3833.01

0.259

5

33.01

18:3n-3-18:3n-3

36

3702.13

0.2748

5

102.13

1:0-24:0 1:0-25:0 1:0-26:0 1:0-27:0 1:0-28:0 1:0-29:0 1:0-30:0 1:0-31:0 1:0-32:0 1:0-33:0 1:0-34:0

25 26 27 28 29 30 31 32 33 34 35

2712.67 2813.71 2915.05 3015.64 3116.12 3216.19 3316.65 3417.17 3518.23 3619.24 3719.84

0.094 0.047 0.093 0.105 0.159 0.085 0.010 0.094 0.146 0.391 0.300

5 4 5 5 5 5 4 5 5 5 5

212.67 213.71 215.05 215.64 216.12 216.19 216.65 217.17 218.23 219.24 219.84

2:0-23:0 2:0-24:0 2:0-25:0 2:0-26:0 2:0-27:0 2:0-28:0 2:0-29:0 2:0-30:0 2:0-31:0 2:0-32:0 2:0-33:0 2:0-34:0

25 26 27 28 29 30 31 32 33 34 35 36

2678.15 2779.13 2880.08 2980.88 3080.81 3180.91 3280.94 3381.50 3482.11 3582.84 3683.54 3782.63

0.263 0.129 0.082 0.113 0.093 0.153 0.146 0.143 0.241 0.180 0.446 0.347

6 6 6 6 5 6 6 6 6 6 3 2

178.15 179.13 180.08 180.88 180.81 180.91 180.94 181.50 182.11 182.84 183.54 182.63

3:0-22:0 3:0-24:0 3:0-26:0 3:0-28:0 3:0-30:0 3:0-31:0 3:0-32:0

25 27 29 31 33 34 35

2675.85 2877.44 3078.35 3278.46 3479.66 3582.44 3681.16

0.125 0.189 0.206 0.078 0.090 0.476 0.396

7 7 7 7 6 5 7

175.85 177.44 178.35 178.46 179.66 182.44 181.16

4:0-22:0 4:0-23:0 4:0-24:0 4:0-25:0 4:0-26:0 4:0-27:0 4:0-28:0 4:0-29:0 4:0-30:0 4:0-31:0 4:0-32:0

26 27 28 29 30 31 32 33 34 35 36

2771.85 2872.77 2973.76 3073.76 3173.94 3273.93 3374.39 3475.19 3575.92 3676.65 3776.57

0.123 0.221 0.131 0.186 0.186 0.163 0.200 0.257 0.167 0.681 0.501

8 8 8 8 8 6 7 8 7 4 4

171.85 172.77 173.76 173.76 173.94 173.93 174.39 175.19 175.92 176.65 176.57

Compound

Number of carbon atoms (NCA)

Kov´ats index (I)

STD

n

Reduced Kov´ats index (RKI)

6:0-20:0 6:0-22:0 6:0-24:0 6:0-26:0

26 28 30 32

2765.80 2966.35 3167.10 3368.55

0.201 0.114 0.164 0.179

5 5 5 5

165.80 166.35 167.10 168.55

8:0-17:0 8:0-20:0 8:0-22:0 8:0-24:0 8:0-25:0 8:0-26:0

25 28 30 32 33 34

2658.84 2959.97 3160.05 3361.03 3461.54 3562.26

0.188 0.438 0.073 0.303 0.513 0.208

7 6 6 7 3 7

158.84 159.97 160.05 161.03 161.54 162.26

10:0-16:0 10:0-17:0 10:0-20:0 10:0-22:0 10:0-24:0 10:0-25:0 10:0-26:0

26 27 30 32 34 35 36

2753.72 2854.03 3153.90 3354.63 3356.31 3656.93 3757.38

0.140 0.325 0.139 0.157 0.150 0.517 0.079

4 3 4 5 5 3 5

153.72 154.03 153.90 154.63 156.31 156.93 157.38

22:0-6:0 23:0-6:0 24:0-6:0 25:0-6:0 26:0-6:0 27:0-6:0 28:0-6:0 30:0-6:0

28 29 30 31 32 33 34 36

2967.79 3068.46 3169.06 3268.87 3369.52 3469.77 3570.57 3772.13

0.040 0.322 0.243 0.321 0.130 0.407 0.253 0.473

7 7 7 7 7 6 7 4

167.79 168.46 169.06 168.87 169.52 169.77 170.57 172.13

23:0-2:0 24:0-2:0 25:0-2:0 26:0-2:0 27:0-2:0 28:0-2:0 29:0-2:0 30:0-2:0 31:0-2:0

25 26 27 28 29 30 31 32 33

2693.69 2794.87 2895.22 2995.60 3095.97 3196.58 3296.78 3398.06 3498.13

0.113 0.059 0.194 0.116 0.129 0.190 0.165 0.086 0.120

5 5 5 5 5 5 5 5 5

193.69 194.87 195.22 195.60 195.97 196.58 196.78 198.06 198.13

and possibly, non-lipid compounds, could be identified. Since higher fatty acids are present in a majority of sources either in the forms of their derivatives (wax esters, sterol esters, triacylglycerols, diacylglycerols or monoacylglycerols) or as free fatty acids, it was often impossible to re-identify the composition of all above-mentioned derivatives. The same principle is true for alcohols and sterols. An advanced development of the separation techniques later enabled to separate the determined groups of lipids and to analyze each separated lipid group by means of the GC and/or GC–MS either in their native state or after their transesterification. Even individual homologues of higher esters turned out to be mixtures of isomers. Analogously, some branched-chain hydrocarbons have similar retention times, and single isomers co-elute [16] and their identification is only possible by a careful evaluation of their mass spectra. Since even much higher esters (up to C60 ) are present in many natural sources, GC and GC–MS are becoming the only applicable analytical methods [8,9,12–14,17–21]. Due to the already mentioned similarity of the retention times of many isomeric esters, difficulties occur during the evaluation of their mass spectra, because a majority of

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Table 2 Higher esters in ascending order of Kov´ats indexes (I) Compound

Number of carbon atoms (NCA)

Kov´ats index (I)

Difference of I

STD

n

Reduced Kov´ats index (RKI)

12:0-12:0 8:0-17:0 3:0-22:0 2:0-23:0 23:0-2:0 1:0-24:0 12:0-14:1n-5 14:1n-5-12:0 12:0-14:0 14:0-12:0 10:0-16:0 6:0-20:0 4:0-22:0 2:0-24:0 24:0-2:0 1:0-25:0 10:0-17:0 4:0-23:0 3:0-24:0 2:0-25:0 25:0-2:0 1:0-26:0 12:0-16:1n-7 16:1n-7-12:0 14:1n-5-14:1n-5 14:1n-5-14:0 14:0-14:0 12:0-16:0 16:0-12:0 8:0-20:0 6:0-22:0 22:0-6:0 4:0-24:0 2:0-26:0 26:0-2:0 1:0-27:0 23:0-6:0 4:0-25:0 3:0-26:0 2:0-27:0 27:0-2:0 1:0-28:0 12:0-18:2n-6 18:2n-6-12:0 14:1n-5-16:1n-7 16:1n-7-14:1n-5 12:0-18:1n-9 12:0-18:3n-3 18:1n-9-12:0 18:3n-3-12:0 14:0-16:1n-7 16:1n-7-14:0 16:0-14:1n-5 14:1n-5-16:0 14:0-16:0 16:0-14:0 12:0-18:0 18:0-12:0 10:0-20:0 8:0-22:0 6:0-24:0 24:0-6:0 4:0-26:0 2:0-28:0

24 25 25 25 25 25 26 26 26 26 26 26 26 26 26 26 27 27 27 27 27 27 28 28 28 28 28 28 28 28 28 28 28 28 28 28 29 29 29 29 29 29 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30

2554.05 2658.84 2675.85 2678.15 2693.69 2712.67 2742.52 2743.36 2751.90 2752.66 2753.72 2765.80 2771.85 2779.13 2794.87 2813.71 2854.03 2872.77 2877.44 2880.08 2895.22 2915.05 2931.17 2932.01 2932.37 2941.70 2950.07 2950.68 2951.62 2959.97 2966.35 2967.79 2973.76 2980.88 2995.60 3015.64 3068.46 3073.76 3078.35 3080.81 3095.97 3116.12 3116.66 3118.21 3121.35 3121.54 3122.55 3123.04 3123.53 3124.42 3129.40 3129.99 3140.65 3140.74 3148.37 3148.72 3150.19 3150.85 3153.90 3160.05 3167.10 3169.06 3173.94 3180.91

104.79 17.01 2.30 15.54 18.98 29.85 0.84 8.54 0.76 1.06 12.08 6.05 7.28 15.74 18.84 40.32 18.74 4.67 2.64 15.14 19.83 16.12 0.84 0.36 9.33 8.37 0.61 0.94 8.35 6.38 1.44 5.97 7.12 14.72 20.04 52.82 5.30 4.59 2.46 15.16 20.15 0.54 1.55 3.14 0.19 1.01 0.49 0.49 0.89 4.98 0.59 10.66 0.09 7.63 0.35 1.47 0.66 3.05 6.15 7.05 1.96 4.88 6.97

0.030 0.188 0.125 0.263 0.113 0.094 0.270 0.172 0.070 0.033 0.140 0.201 0.123 0.129 0.059 0.047 0.325 0.221 0.189 0.082 0.194 0.093 0.109 0.028 0.108 0.111 0.172 0.034 0.170 0.438 0.114 0.040 0.131 0.113 0.116 0.105 0.322 0.186 0.206 0.093 0.129 0.159 0.155 0.270 0.145 0.124 0.161 0.074 0.075 0.166 0.170 0.066 0.017 0.136 0.126 0.158 0.103 0.212 0.139 0.073 0.164 0.243 0.186 0.153

6 7 7 6 5 5 5 5 5 6 4 5 8 6 5 4 3 8 7 6 5 5 5 4 6 5 5 4 6 6 5 7 8 6 5 5 7 8 7 5 5 5 5 5 5 6 5 6 5 5 5 4 4 5 5 5 5 6 4 6 5 7 8 6

154.05 158.84 175.85 178.15 193.69 212.67 142.52 143.36 151.90 152.66 153.72 165.80 171.85 179.13 194.87 213.71 154.03 172.77 177.44 180.08 195.22 215.05 131.17 132.01 132.37 141.70 150.07 150.68 151.62 159.97 166.35 167.79 173.76 180.88 195.60 215.64 168.46 173.76 178.35 180.81 195.97 216.12 116.66 118.21 121.35 121.54 122.55 123.04 123.53 124.42 129.40 129.99 140.65 140.74 148.37 148.72 150.19 150.85 153.90 160.05 167.10 169.06 173.94 180.91

K. Str´ansk´y et al. / J. Chromatogr. A 1128 (2006) 208–219

213

Table 2 (Continued ) Compound

Number of carbon atoms (NCA)

Kov´ats index (I)

28:0-2:0 18:1n-9-14:1n-5 1:0-29:0 12:0-20:4n-6 25:0-6:0 4:0-27:0 3:0-28:0 2:0-29:0 29:0-2:0 14:1n-5-18:2n-6 18:2n-6-14:1n-5 16:1n-7-16:1n-7 14:1n-5-18:1n-9 14:1n-5-18:3n-3 14:0-18:2n-6 18:3n-3-14:1n-5 1:0-30:0 18:2n-6-14:0 14:0-18:1n-9 18:1n-9-14:0 14:0-18:3n-3 18:3n-3-14:0 16:0-16:1n-7 16:1n-7-16:0 14:1n-5-18:0 18:0-14:1n-5 16:0-16:0 14:0-18:0 18:0-14:0 12:0-20:0 20:0-12:0 10:0-22:0 10:0-24:0 8:0-24:0 6:0-26:0 26:0-6:0 4:0-28:0 2:0-30:0 30:0-2:0 1:0-31:0 14:1n-5-20:4n-6 14:0-20:4n-6 8:0-25:0 27:0-6:0 4:0-29:0 3:0-30:0 2:0-31:0 16:1n-7-18:2n-6 18:2n-6-16:1n-7 31:0-2:0 16:1n-7-18:1n-9 18:1n-9-16:1n-7 16:1n-7-18:3n-3 18:3n-3-16:1n-7 16:0-18:2n-6 18:2n-6-16:0 1:0-32:0 16:0-18:1n-9 18:1n-9-16:0 16:0-18:3n-3 18:3n-3-16:0 16:1n-7-18:0 18:0-16:1n-7 14:1n-5-20:0 20:0-14:1n-5

30 32 30 32 31 31 31 31 31 32 32 32 32 32 32 32 31 32 32 32 32 32 32 32 32 32 32 32 32 32 32 32 34 32 32 32 32 32 32 32 34 34 33 33 33 33 33 34 34 33 34 34 34 34 34 34 33 34 34 34 34 34 34 34 34

3196.58 3213.80 3216.19 3259.96 3268.87 3273.93 3278.46 3280.94 3296.78 3307.85 3308.64 3310.31 3314.12 3314.99 3315.74 3315.81 3316.65 3316.90 3321.09 3321.79 3322.43 3323.48 3328.75 3328.91 3340.46 3340.55 3347.13 3347.94 3348.04 3350.36 3351.81 3354.63 3356.31 3361.03 3368.55 3369.52 3374.39 3381.50 3398.06 3417.17 3452.29 3459.60 3461.54 3469.77 3475.19 3479.66 3482.11 3497.40 3497.96 3498.13 3502.79 3503.04 3504.89 3505.81 3515.52 3516.49 3518.23 3520.42 3520.87 3522.64 3523.60 3528.92 3528.96 3541.84 3542.10

Difference of I 15.67 17.22 2.39 43.77 8.91 5.06 4.53 2.48 15.84 11.07 0.79 1.67 3.81 0.87 0.75 0.07 0.84 0.25 4.19 0.70 0.64 1.05 5.27 0.16 11.55 0.09 6.58 0.81 0.10 2.32 1.45 2.82 1.68 4.72 7.52 0.97 4.87 7.11 16.56 19.11 35.12 7.31 1.94 8.23 5.42 4.47 2.45 15.29 0.56 0.17 4.66 0.25 1.85 0.92 9.71 0.97 1.74 2.19 0.45 1.77 0.96 5.32 0.04 12.88 0.26

STD

n

Reduced Kov´ats index (RKI)

0.190 0.063 0.085 0.242 0.321 0.163 0.078 0.146 0.165 0.176 0.1447 0.112 0.185 0.022 0.106 0.182 0.010 0.261 0.103 0.067 0.149 0.064 0.139 0.149 0.119 0.145 0.112 0.033 0.034 0.192 0.219 0.157 0.150 0.303 0.179 0.130 0.200 0.143 0.086 0.094 0.050 0.182 0.513 0.407 0.257 0.090 0.241 0.151 0.128 0.120 0.125 0.157 0.146 0.153 0.114 0.140 0.146 0.248 0.123 0.082 0.050 0.084 0.067 0.166 0.179

5 5 5 7 7 6 7 6 5 5 5 5 5 4 5 5 4 5 5 5 6 5 5 5 5 5 5 4 4 6 6 5 5 7 5 7 7 6 5 5 4 7 3 6 8 6 6 5 5 5 5 5 5 5 5 5 5 5 5 6 4 5 5 5 5

196.58 113.80 216.19 59.96 168.87 173.93 178.46 180.94 196.78 107.85 108.64 110.31 114.12 114.99 115.74 115.81 216.65 116.90 121.09 121.79 122.43 123.48 128.75 128.91 140.46 140.55 147.13 147.94 148.04 150.36 151.81 154.63 156.31 161.03 168.55 169.52 174.39 181.50 198.06 217.17 52.29 59.60 161.54 169.77 175.19 179.66 182.11 97.40 97.96 198.13 102.79 103.04 104.89 105.81 115.52 116.49 218.23 120.42 120.87 122.64 123.60 128.92 128.96 141.84 142.10

214

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Table 2 (Continued ) Compound

Number of carbon atoms (NCA)

Kov´ats index (I)

Difference of I

STD

n

Reduced Kov´ats index (RKI)

16:0-18:0 18:0-16:0 14:0-20:0 20:0-14:0 12:0-22:0 22:0-12:0 8:0-26:0 28:0-6:0 4:0-30:0 3:0-31:0 2:0-32:0 1:0-33:0 16:1n-7-20:4n-6 10:0-25:0 16:0-20:4n-6 4:0-31:0 3:0-32:0 2:0-33:0 18:2n-6-18:2n-6 18:1n-9-18:2n-6 18:2n-6-18:1n-9 18:2n-6-18:3n-3 18:3n-3-18:2n-6 18:1n-9-18:1n-9 18:1n-9-18:3n-3 18:3n-3-18:1n-9 18:3n-3-18:3n-3 18:0-18:2n-6 18:2n-6-18:0 1:0-34:0 18:0-18:1n-9 18:1n-9-18:0 18:0-18:3n-3 18:3n-3-18:0 16:1n-7-20:0 20:0-16:1n-7 14:1n-5-22:0 22:0-14:1n-5 18:0-18:0 16:0-20:0 20:0-16:0 14:0-22:0 22:0-14:0 10:0-26:0 30:0-6:0 4:0-32:0 2:0-34:0 18:2n-6-20:4n-6 18:1n-9-20:4n-6 18:0-20:4n-6 20:0-18:2n-6 18:2n-6-20:0 20:0-18:1n-9 18:1n-9-20:0 20:0-18:3n-3 18:3n-3-20:0 16:1n-7-22:0 22:0-16:1n-7 18:0-20:0 20:0-18:0 16:0-22:0 22:0-16:0 20:0-20:4n-6 22:0-18:2n-6 18:2n-6-22:0

34 34 34 34 34 34 34 34 34 34 34 34 36 35 36 35 35 35 36 36 36 36 36 36 36 36 36 36 36 35 36 36 36 36 36 36 36 36 36 36 36 36 36 36 36 36 36 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 40 40 40

3546.45 3546.52 3547.92 3548.72 3550.68 3552.51 3562.26 3570.57 3575.92 3582.44 3582.84 3619.24 3642.73 3656.93 3660.65 3676.65 3681.16 3683.54 3685.73 3690.81 3691.23 3693.85 3694.05 3695.89 3698.65 3699.58 3702.13 3716.27 3717.06 3719.84 3720.44 3720.72 3723.93 3724.49 3730.26 3730.31 3743.34 3743.66 3745.68 3746.49 3747.02 3748.29 3749.34 3757.38 3772.13 3776.57 3782.63 3833.01 3837.23 3863.07 3918.43 3919.22 3922.10 3922.41 3926.60 3927.21 3931.84 3932.05 3945.99 3946.15 3946.89 3947.81 4066.48 4120.82 4121.72

4.35 0.07 1.40 0.80 1.96 1.83 9.75 8.31 5.35 6.52 0.40 3.64 23.49 14.20 3.72 16.00 4.51 2.38 2.19 5.08 0.42 2.62 0.20 1.84 2.76 0.93 2.55 14.14 0.79 2.78 0.60 0.28 3.21 0.56 5.77 0.05 13.03 0.32 2.02 0.81 0.53 1.27 1.05 8.04 14.75 4.44 6.06 50.38 4.22 25.84 55.36 0.79 2.88 0.31 4.19 0.61 4.63 0.21 13.94 0.16 0.74 0.92 118.67 54.34 0.90

0.091 0.319 0.131 0.183 0.252 0.122 0.208 0.253 0.167 0.476 0.180 0.391 0.253 0.517 0.271 0.681 0.396 0.446 0.117 0.191 0.090 0.298 0.085 0.327 0.116 0.127 0.2748 0.046 0.194 0.300 0.127 0.181 0.106 0.051 0.143 0.086 0.259 0.098 0.017 0.064 0.193 0.109 0.247 0.079 0.473 0.501 0.347 0.259 0.245 0.177 0.134 0.273 0.233 0.116 0.179 0.198 0.289 0.076 0.178 0.056 0.046 0.142 0.295 0.367 0.218

5 5 6 5 6 6 7 7 7 5 6 5 5 3 7 4 7 3 5 5 4 5 4 5 5 5 5 5 5 5 5 5 6 4 5 5 5 5 4 6 5 6 5 5 4 4 2 5 5 7 5 5 5 5 5 5 5 4 6 4 5 5 7 5 5

146.45 146.52 147.92 148.72 150.68 152.51 162.26 170.57 175.92 182.44 182.84 219.24 42.73 156.93 60.65 176.65 181.16 183.54 85.73 90.81 91.23 93.85 94.05 95.89 98.65 99.58 102.13 116.27 117.06 219.84 120.44 120.72 123.93 124.49 130.26 130.31 143.34 143.66 145.68 146.49 147.02 148.29 149.74 157.38 172.13 176.57 182.63 33.01 37.33 63.07 118.43 119.22 122.10 122.41 126.60 127.21 131.84 132.05 145.99 146.15 146.89 147.81 66.48 120.82 121.72

K. Str´ansk´y et al. / J. Chromatogr. A 1128 (2006) 208–219

215

Table 2 (Continued ) Compound

Number of carbon atoms (NCA)

Kov´ats index (I)

Difference of I

STD

n

Reduced Kov´ats index (RKI)

22:0-18:1n-9 18:1n-9-22:0 22:0-18:3n-3 18:3n-3-22:0 20:0-20:0 18:0-22:0 22:0-18:0 22:0-20:4n-6 20:0-22:0 22:0-20:0 22:0-22:0

40 40 40 40 40 40 40 42 42 42 44

4124.07 4124.32 4129.59 4130.13 4146.16 4146.31 4146.65 4270.34 4346.38 4346.67 4547.32

2.35 0.25 5.27 0.54 16.03 0.15 0.34 123.69 76.04 0.29 200.65

0.079 0.184 0.328 0.132 0.118 0.128 0.269 0.369 0.200 0.151 0.080

4 5 5 5 6 6 5 6 6 6 5

124.07 124.32 129.59 130.13 146.16 146.31 146.65 70.34 146.38 146.67 147.32

Table 3 Parameters of the equation f = y0 + a × x for groups of esters from Fig. 6 Group of esters

Coefficient y0

Std error

Coefficient a

Std error

Coefficient of correlation R

Methyl Ethyl Propyl Butyl Hexyl Octyl Decyl

197.0191 168.6984 164.2067 160.6463 152.5060 149.8484 143.7075

1.2194 1.3007 1.5417 1.1550 2.9793 1.2681 2.1406

0.6464 0.4075 0.4760 0.4446 0.5010 0.3549 0.3679

0.0404 0.0424 0.0511 0.0371 0.1024 0.0416 0.0677

0.9829 0.9500 0.9778 0.9701 0.9607 0.9736 0.9249

the chromatographic peaks represent mixtures of non-separated compounds. Our effort was directed to obtaining precise and reproducible retention data from as many as possible reference compounds of higher esters. Due to a relatively high number of carbon atoms and thus high boiling points of the compounds studied, a DB-1 ms column was chosen, which is commonly used as a non-polar column with a maximum working temperature up to 340/360 ◦ C and with a very low bleed characteristics, suitable for GC–MS measurements. To be able to analyze long homologue series of compounds

during a single chromatographic run, one must use a temperature program. A slow temperature program 1 ◦ C/min starting at 240 ◦ C has been found the most suitable. Hydrogen was used as a carrier gas, and its increased flow velocity (u¯ = 60 cm/s) was applied. Under employing the maximum working temperature at temperature program (360 ◦ C), a GC analysis of n-alkanes up to C54 and esters up to C52 –C53 can be performed. According the original formula [22] only two n-alkanes are used for standard calculation of the Kov´ats retention indexes (I). The interpolation line is then determined by these two points

Table 4 Dependence of retention times (RT) and of Kov´ats indexes (I) on number of working hours of the DB-1 ms column Compound

Number of carbon atoms (NCA)

RT (min)

Min

%

FEB 7

JUL 7a

I

%

4.862 7.503 11.450 16.890 23.777 31.780

0.354 0.544 0.787 1.069 1.338 1.555

6.79 6.76 6.43 5.95 5.32 4.66

2554.05 2752.66 2951.62 3150.85 3351.81 3552.51

2553.85 2752.44 2951.58 3150.70 3351.63 3552.02

0.20 0.22 0.04 0.15 0.18 0.49

0.008 0.008 0.001 0.005 0.005 0.014

5.216 8.047 12.237 17.959 25.115 33.335

Compound

Number of carbon atoms (NCA)

RT (min)

a b

Difference

JUL 7a

24 26 28 30 32 34

32 34 36

I

FEB 7

12:0-12:0 14:0-12:0 16:0-12:0 18:0-12:0 20:0-12:0 22:0-12:0

14:1n-5-18:3n-3 16:1n-7-18:3n-3 18:1n-9-18:3n-3

Difference

Difference 7b

APR 22

JUL

23.058 30.577 38.937

22.374 29.766 38.005

Number of working hours from FEB 7 to JUL 7: 600 h. Number of working hours from APR 22 to JUL 7: 250 h.

I

Difference 7b

Min

%

APR 22

JUL

0.682 0.811 0.932

2.96 2.65 2.39

3314.99 3504.89 3698.65

3314.43 3504.43 3698.16

I

%

0.56 0.46 0.49

0.017 0.013 0.013

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K. Str´ansk´y et al. / J. Chromatogr. A 1128 (2006) 208–219

only; however, even a minor deviation of the retention time of either of these n-alkanes results in affecting of a slope of the interpolation line, and affects the calculated values of the retention index. Using a more exact measurement, it was later found that the dependence of logarithm of the retention time on the number of carbon atom of members of homologous series is not linear (first order polynomial) but it can be described by higher order polynomial much more accurately [23,24]. Higher accuracy of calculation is enabled by using more than two reference n-alkanes for the analysis. The advantage of this approach results in obtaining more experimental points, through which a curve of the higher polynomial may be interpolated more exactly, and the I values calculated are also more exact and more reproducible. The program used for calculation of the Kov´ats retention indexes, was developed earlier for the calculation of the ECL values of methyl esters of fatty acids [15], and it enables to measure and to analyze several compounds in one chromatographic run simultaneously [25]. Moreover, an advantage of a selection of either even n-alkanes or odd n-alkanes can be used, when the peak(s) of the studied compound(s) coincide(s) during the chromatography with the peak(s) of the co-injected n-alkanes, or when the separation of the peaks mentioned does not reach the baseline, when the apexes of the studied compound and the n-alkane are influenced. A sufficient condition for application of higher polynomials consists in a number of reference n-alkanes, which has to be at least a double of the degree of applied polynomial. To achieve separation of two different esters to a base line, a difference in the values of their retention indexes has to be at least 10 index units. If this value is lower than 10, either a pair of non-separated peaks or a single peak occurs (Fig. 1). It is obvious from almost all graphs and from Tables 1 and 2 that both a number of isomeric esters and esters of different groups cannot be separated by GC. If they are present in the studied samples, mixed mass spectra should be expected during the GC–MS analyses. Since the Kov´ats indexes (I) are relatively great values, due to multiplying by factor 100, we use reduced Kov´ats indexes (RKI), the equation for calculation of which is RKI = I − 100 × NCA, where NCA means number of carbon atoms. RKI values are analogous to the fractional chain length (FCL) values [26] and express the influence of the presence and position of the ester functionality or position of double bond(s) on the chromatographic stationary phase in more details, and moreover, are much more convenient for construction of graphs (Table 1). Based on the values given in Table 1, several types of graphs could be constructed. Fig. 2 shows dependence of the RKI on NCA in the alcohol moiety in the C32 ester. The shortest retention time was found for the compound bearing its ester functionality in the middle of the chain. This finding relates both to the steric structure of the ester molecule and thus to the ester boiling point. Additional graphs summarize dependence of the retention data, given by the dependence of the RKI values on a NCA in several series of related esters. Fig. 3a shows the above-described dependence calculated for 6 series of esters, the chains of which are formed from different number of carbon atoms in the alco-

Fig. 1. Gas chromatograms of pairs of individual esters: (a) 18:018:0 (I1 = 3745.68) and 22:0-14:0 (I2 = 3749.34): (I2 − I1 = 3.66); (b) 16:018:0 (I1 = 3646.35) and 22:0-12:0 (I2 = 3552.51): (I2 − I1 = 6.6); (c) 14:1n-520:0 (I1 = 3541.84) and 22:0-12:0 (I2 = 3552.51): (I2 − I1 = 10.67); (d) 18:018:3 (I1 = 3723.93) and 18:0-18:0 (I2 = 3745.68): (I2 − I1 = 21.75).

hol moiety of esters (from C12 to C22 ). Fig. 3b shows similar dependence for isomeric esters, in which the number of carbon atoms varies in the acid moiety (from C12 to C22 ). Fig. 4a and b show dependence of retention data for pairs of esters, which display identical composition either in their

Fig. 2. Dependence of reduced Kov´ats index (RKI) values on number of carbon atoms (NCA) in alcohol moiety of C32 esters.

K. Str´ansk´y et al. / J. Chromatogr. A 1128 (2006) 208–219

Fig. 3. Dependences of RKI values on NCA of esters (six homologous series, six even esters in each series): (a) different NCA in alcohol moieties; (b) different NCA in acid moieties; for information about individual esters see legend under the picture.

217

Fig. 4. Dependences of RKI values on NCA of: (a) esters (three pairs of homologous series of esters, six even esters in each series) with identical composition either in acid moiety or in alcohol moiety; (b) two pairs of homologous series of another esters (always three members); for information about individual esters see legend under the picture.

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acid moiety or in their alcohol moiety. It clearly follows from graphical dependence that such ester pairs cannot be separated. Fig. 5a shows the influence of a number of double bonds in the ester molecule on chromatographic behavior in comparison with the corresponding saturated ester. The longest retention times have saturated esters. The retention values do not shorten, however, according to the increasing number of double bonds. The esters bearing three double bonds in the alcohol moiety are eluted before the esters bearing one or two double bonds. Similar chromatographic behavior can be found with esters bearing one, two, or three double bond(s) in the acid moiety (Fig. 5b). Fig. 6 displays comparison of dependences of retention data of methyl, ethyl, propyl, butyl, hexyl, octyl, and decyl esters on NCA. Dependence was found to be linear in the range of chain

Fig. 6. Dependences of RKI values on NCA of methyl, ethyl, propyl, butyl, hexyl, octyl, and decyl esters (even and odd members from C25 to C36 ); for information about individual esters and about linearity and parallelism see legend under the picture and Table 3.

lengths from C25 to C36 and the lines interpolated through the points are almost parallel (with exclusion of methyl esters) (cf. also Table 3). Compounds displaying high boiling point give often tailing peaks in comparison with the volatile compounds. In spite of this fact, the Kov´ats retention indexes (I) were calculated with accuracy lower than 0.5 unit. An average STD value, based on the given 1105 analyses (after excluding outliers) was found to be 0.285. An electronic regulation of the carrier gas velocity could also improve reproducibility of the analyses. Moreover, with application of a constant carrier gas velocity during whole temperature gradient program, analyzing of samples containing compounds with even higher number of carbon atoms could be possible. The whole set of analyses took about 600 working hours, in which time the new DB-1 ms column was repeatedly subjected to the temperature gradient program from 240 to 340 ◦ C. It resulted in shortening of the retention times due to bleeding of the column. However, the difference among the retention indexes of the corresponding saturated ester is 0.21 in average only (Table 4). Evaluating the esters with four double bonds in the molecules, this difference is 0.51 in average. Thus, the retention times are affected by a long-term use of a DB-1 ms column; however, the values of the retention indexes are affected only negligibly (thanks to a careful purification of the carrier gas before its entering the column, see Section 2).

Fig. 5. Dependences of RKI values on NCA of saturated and unsaturated esters (four homologous series, six even esters in each series); (a) one, two or three double bonds in alcohol moiety; (b) one, two or three double bonds in acid moiety; for information about individual esters see legend under the picture.

4. Conclusions Even if a series of more than 200 wax esters was available for this study, it still represents a negligible number of theoreti-

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cally available compounds, which can be found in natural lipid sources, and which have not been identified yet in full details or not at all. Ester homologues with odd number of carbon atoms and homologues with different combination of double bonds both in the acid moiety and in the alcohol moiety could not be analyzed for their unavailability. Our study however brings new and valuable chromatographic data that contribute to the knowledge on chromatographic behavior of higher esters in general. Thus, our results will enable other authors to identify unknown esters in natural sources more easily than before based on Kov´ats retention indexes. Acknowledgements The financial support from the Grant Agency of the Czech Republic (203/04/0120) and the Grant Agency of the Academy of Sciences of the Czech Republic (A4055403) is herewith acknowledged with appreciation. The authors are highly indebted to Dr. Milan Streibl for offering samples of synthetic higher esters, which are not available commercially, to Mrs. Helena Ernyeiov´a for her skilful technical assistance and for calculation of the Kov´ats retention indexes, and to Mrs. Anna Nekolov´a for her assistance with the GC–MS analyses. References [1] A.H. Warth, The Chemistry and Technology of Waxes, 2nd ed., Reinhold, New York, 1956. [2] P.E. Kolattukudy (Ed.), Chemistry and Biochemistry of Natural Waxes, Elsevier, Oxford, 1976.

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[3] S. Misra, A. Ghosh, in: H.F. Linskens, J.F. Jackson (Eds.), Essential Oils and Waxes, Springer, Berlin, 1991, p. 205. [4] D.W. Stanley-Samuelson, D.R. Nelson (Eds.), Insect Lipids: Chemistry, Biochemistry and Biology, University of Nebraska Press, Lincoln and London, 1993. [5] R.J. Hamilton (Ed.), Waxes: Chemistry, Molecular Biology and Functions, Oily Press, Dundee, 1995. [6] P.J. Weldon, A. Shafagati, J.W. Wheeler, Lipids 23 (1988) 727. [7] M.H.A. Dekker, T. Piersma, J.S.S. Damst´e, Lipids 35 (2000) 533. [8] G.F. Spencer, J. Am. Oil Chem. Soc. 56 (1979) 642. [9] T. Horiguchi, Y. Takase, Y. Arai, H. Ageta, Nat. Med. (Tokyo) 53 (1999) 105. [10] J.R. Sargent, R.R. Gatten, R.J. Henderson, Pure Appl. Chem. 53 (1981) 867. [11] R.G. Ackman (Ed.), Marine Biogenic Lipids, Fats, and Oils, CRC Press, Boca Raton, 1989. [12] G. Kattner, M. Graeve, W. Ernst, J. Chromatogr. 513 (1990) 327. [13] M. Graeve, G. Kattner, Mar. Chem. 39 (1992) 269. [14] S.G. Wakeham, Geochim. Cosmochim. Acta 46 (1982) 2239. [15] K. Str´ansk´y, T. Jurs´ık, A. V´ıtek, J. Skoˇrepa, J. High Resolut. Chromatogr. 15 (1992) 730. [16] D.A. Carlson, U.R. Bernier, B.D. Sutton, J. Chem. Ecol. 24 (1998) 1845. [17] S.G. Wakeham, N.M. Frew, Lipids 17 (1982) 831. [18] Y. Itabashi, T. Takagi, J. Chromatogr. 299 (1984) 351. ˇ [19] T. Rezanka, M. Podojil, J. Chromatogr. 362 (1986) 399. [20] W.W. Christie, Gas Chromatography and Lipids. A Practical Guide, Oily Press, Ayr, 1989, p. 218. ˇ [21] W.M. Dembitsky, T. Rezanka, Phytochemistry 42 (1996) 1075. [22] E. Kov´ats, Helv. Chim. Acta 41 (1958) 1915. [23] G.J. Nelson, Lipids 9 (1974) 254. [24] F.J. Heeg, R. Zinburg, H.-J. Neu, K. Ballschmitter, Chromatographia 12 (1979) 451. [25] I.G. Zenkevich, B.V. Ioffe, J. Chromatogr. A 439 (1988) 185. [26] F.D. Gunstone, B.G. Hersl¨of, Lipid Glossary 2, Oily Press, Bridgwater, 2000, p. 84.