Downloaded via SAN FRANCISCO STATE UNIV on August 6, 2018 at 01:26:15 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
tant, it does not appear as the major air blowing reaction. Smith and Schweyer assumed a high value for the formation of direct carbon to carbon bonds of —94 kcal/mol and demoted their effect where these bonds may in fact vary from -60 to -100 kcal/mol. Weak C-C bonds are more likely to be formed as the free radical initiation reaction will begin with the rupture of the most reactive, i.e., the weakest C-H bond with energies down to —70 kcal/mol. As the energy for the C-H bond rupture rises, the initiation reaction becomes more endothermic compensating for the higher C-C bond strengths. The slight decrease in the heat of reaction as the blowing temperature is increased, is accounted for by increased decarboxylation and a higher dehydrogenation rate. Air Blowing Reaction Mechanism. The primary reaction at low blowing temperatures appears to be polymerization through the paraffinic chain structure with some internal cross-linking and dehydrogenation occurring to increase the relative amounts of naphthenic and aromatic carbon from that of the short residue. The amount of dehydrogenation is small as is dealkylation. As the blowing temperature is increased, more dehydrogenation and dealkylation occur with polymerization lessening. The effect of aromatization of the naphthenic structure is shown by the formation of high unit sheet weights. Since the naphthenic structures appear to be less highly condensed than the aromatic structure, their dehydrogenation will produce unit sheet weights which will no longer conform with the peri-condensed aromatic model. This leads to high values of the unit sheet weight. Diels-Alder reactions where an aromatic ring undergoes a condensation reaction with a dehydrogenated naphthenic ring have a similar but more pronounced effect on the unit sheet weight.
ACKNOWLEDGMENT The author thanks D. F. Orchard, E. J. Dickinson, and N. W. West for their helpful criticism and assistance.
LITERATURE CITED (1) Y. Ichikawa, J. Soc. Chem. Ind., Jpn Suppl. Binding, 39, 405 (1936). (2) A. M. Challón, R. N. Traxler, and J. W. Romberg, Ind. Eng. Chem., 51,
1353 (1959). (3) P. Zakar, R. Csikos, G. Mozes, and M. Kristof, Magy Kem. Lapja, 18 (4), 157 (1963). (4) S. H. Greenfeld, Ind. Eng. Chem., Prod. Res. Dev., 3, 158 (1964). (5) E. J. Dickinson, Proc. Assoc. Asphalt Paving Techno!., 43, 132 (1974). (6) H. Senolt, Bitumen, Teere, Asphaite, Peche, 20 (12), 574 (1969). (7) Osterreichische Mlneralolverwaltung A.G., Brit. Chem. Eng., 14 (11), 9
(1969). (8) J. M. Goppel and J. Knotnerus, 4th World Pet. Congr. Proc., Sec. Ill/G,
399 (1955).
(9) J. Knotnerus, J. Inst. Pet. London, 42, 355 (1956). (10) J. Knotnerus, Erdoel Kohle, 23, 341 (1970). (11) P. G. Campbell and J. R. Wright, J. Res. Natl. Bur. Stand., 68c, 115
(1964). (12) S. M. Dorrence, F. A. Barbour, and J. C. Petersen, Anal. Chem., 46, 2242 (1974). (13) J. C. Petersen, F. A. Barbour, and S. M. Dorrence, Anal. Chem., 47, 107 (1975). (14) D. B. Smith and . E. Schweyer, Ind. Eng. Chem., Proc. Des. Dev., 2, 209 (1963). (15) D. B. Smith and . E. Schweyer, Hydrocarbon Process., 46, 167 (1967). G. A. Haley, Anal. Chem., 44, 580 (1972). (16) (17) K. H. Altgelt, Makromol. Chem., 88, 75 (1965). (18) J. G. Hendrickson and J. C. Moore, J. Polym. Scl., Part A, 1 (4), 167 (1966). (19) K. H. Altgelt, Bitumen, Teere, Asphaite, Peche, 21 (11), 475 (1970). (20) . H. Oelert and J. H. Weber, Erdoel Kohle, 23, 484 (1970). (21) . H. Oelert, D. R. Latham, and W. E. Haines, Sep. Scl., 5, 657 (1970). (22) P. J. Flory, "Principles of Polymer Chemistry", Cornell University Press, Ithaca, N.Y., 1953, p 145. (23) G. A. Haley, Anal. Chem., 43, 371 (1971).
Received for review April 25, 1975. Accepted August 25, 1975. This project was sponsored by the Australian Road Research Board.
Separation of Long Chain Fatty Acids as Phenacyl Esters by High Pressure Liquid Chromatography Richard F. Borch Department of Chemistry, University of Minnesota, Minneapolis, Minn. 55455
The separation and analysis of long chain fatty acid mix-
tures has been applied extensively to obtain information from a number of biological systems. Analytical methods frequently used include gas chromatographic analysis of
methyl (7), benzyl (2), pentafluorobenzyl (3), and p-bromophenacyl (4) esters. More recently, high pressure liquid chromatography (HPLC) has been employed for these separations (5). The preparation of UV-absorbing derivatives has been essential to obtain the sensitivity required for samples in the nanogram range. These derivatives include the benzyl (6), p-nitrobenzyl (7), and 2-naphthacyl (8) esters. We describe here the HPLC analysis of C12-C24 fatty acids as their phenacyl esters. The use of a 10-µ particle size reverse phase column packing provides a high degree
of resolution for acids.
a
number of difficult-to-separate fatty
EXPERIMENTAL Reagents. Acetonitrile was purchased from Burdick & Jackson laboratories, Muskegon, Mich., and used without further purification. Water was distilled from a glass still. Phenacyl bromide was obtained from Aldrich Chemical Company and was recrystallized from pentane. Triethylamine was distilled before use. Stock solutions of phenacyl bromide (12 mg/ml) and triethylamine (10 mg/ ml) in acetone were prepared and stored at 0 °C. Fatty acids were purchased from Sigma Chemical Company. Derivatization Procedure (9). Approximately 100 µ§ of fatty acid, 10 µ of phenacyl bromide solution, and 10 µ of triethylamine solution were combined and allowed to stand overnight at room temperature. Rate of conversion was as follows: 2 hr, 50%; 6 hr, 80%; 8 hr, >90%. Alternatively, complete conversion may be achieved by heating at 50 °C for 2 hr. An aliquot of this solution was injected directly into the liquid chromatograph. Chromatographic Procedure. Analyses were carried out using a Waters Associates Model ALC-100 chromatograph equipped with a Waters Model 660 Solvent Programmer and a Waters UV .
ANALYTICAL CHEMISTRY, VOL. 47, NO. 14, DECEMBER 1975
·
2437
Elution volume, ml.
Figure 1. HPLC of fatty acid phenacyl esters 1, lauric (12:0); 2, myristoleic (14:1); 3, a- and 7-linolenic (18:3); 4, myristic (14:0); 5, palmitoleic (16:1); 6, arachidonic (20:4); 7, frans-palmitoleic (trans 16:1); 8, linoleic (18:2); 9, pentadecanolc (15:0); 10, linolelaidic (trans 18:2); 11, eicosatrienoic (20:3); 12, palmitic (16:0); 13, oleic (18:1, 9), and vaccenic (18: 1, 11); 14, petroselinic (18:1, 6); 15, elaldic (trans 18:1); 16, eicosadienoic (20:2, 11·14); 17, heptadecanoic (17:0); 18, stearic (18:0); 19, elcosaenoic (20:1, 11); 20, nonadecanoic (19:0); 21, arachidic (20:0) and erucic (22:1); 22, heneicosanoic (21:0); 23, behenic (22:0) and nervonic (24:1); 24, lignoceric (24:0). Column 90 cm X 0.64 cm µ-Bondapak C-18; eluent acetonltrlle-water; flow rate 2.0 ml/mln
Peak
detector measuring absorbance at 254 nm. The column was 90 cm X 0.64 cm µ-Bondapak C-18 column (Waters Associates). Acetonitrile:water served as the eluent and was programmed from 67:33 to
97:3 (Figure 1) or from 80:20 to 100:0 (Figure 2) in composition. Flow rate was set at 2.0 ml/min.
RESULTS AND DISCUSSION The phenacyl esters were easily prepared and proved satisfactory for analysis. Residual acetone and phenacyl bromide eluted in the first 20 ml, thus obviating the need for purification prior to analysis. The esters had Xmax = nm (t 14000); at 254 nm, the e decreased to 6000. With the Waters detector, this provided a lower limit of detection of approximately 100 ng of C-18 acid. Use of a variable-wavelength detector of greater sensitivity should decrease this detection limit to ~10 ng for these derivatives. Excellent chromatographic properties were observed for the phenacyl esters using the µ-Bondapak C-18 column and acetonitrile-water as eluent (see Figure 1). Although the p-nitrobenzyl esters (7) showed greater molar absorption at 254 nm, the resolution of these derivatives was significantly reduced as compared with the phenacyl esters. Acetonitrile-water also provided resolution superior to metha-
nm)
(254
Absorbance
nol-water
Elution volume, ml.
Figure 2. HPLC of phenacyl esters 1, lauric (12:0); 2, myristoleic (14:1); 3, llnolenic (18:3); 4, myristic (14: 0); 5, arachidonic (20:4); 6, linoleic (18:2); 7, eicosatrienoic (20:3); 8, palmitic (16:0); 9, oleic (18:1, 9); 10, petroselinic (18:1, 6); 11, eicosadienoic (20: 2); 12, stearic (18:0); 13, arachidic (20:0); 14, behenic (22:0); 15, lignoceric
Peak
(24:0). Column 90 cm X 0.64 cm µ-Bondapak C-18; eluent acetonltrllewater; flow rate 2.0 ml/mln
2438
·
or
tetrahydrofuran-water. Although
a
“continu-
ous-change” solvent program was found which gave equivalent separations, the “step-change program” is illustrated here because solvent changes can be carried out manually without an automatic programmer. Several characteristics of this separation should be noted. First, separation by chain length alone is dramatic, the retention volume increasing with increasing chain length. Note for example, in Figure 1, the separation of the 14:0, 15:0, 16:0, 17:0, and 18:0 acids represented by peaks 3, 9, 12, 17, 18, respectively. Second, increasing unsaturation decreases the retention volume of the phenacyl esters. Thus the C-20 fatty acids 20:0, 20:1, 20:2, 20:3, and 20:4 are represented in Figure 1 by peaks 21, 19, 16, 11, and 6, respectively. Third, the trans unsaturated fatty acid derivatives are intermediate in retention volume between the sat-
ANALYTICAL CHEMISTRY, VOL. 47, NO. 14, DECEMBER 1975
urated and the cis unsaturated derivatives, as expected on the basis of conformational changes induced by introduction of the respective double bonds. Peaks 4, 13, 21, and 23 each represent a mixture of two fatty acid derivatives. Peak 23 can be resolved by omitting the solvent change to 97:3 acetonitrile-water and completing the separation using 80:20 acetonitrile-water. This is not done routinely because it prolongs the analysis time considerably. Should it be essential to resolve peaks 4, 13, and 21, the desired peak is collected from the chromatograph and re-injected in a separate analysis using the “recycle” mode of chromatograph. When applying this method to the analysis of fatty acids derived from a particular biological species, we often find it unnecessary to use the extensive separation illustrated in Figure 1. Consideration of the commonly occurring natural fatty acids (Peaks 1-6, 8, 12, 13, 16, 18, 19, 21, 23, and 24 in Figure 1) suggests that a faster separation with lower resolution might be adequate. The key elements in such an analysis are palmitoleic and arachidonic acids (peaks 5 and 6, Figure 1). Complete resolution of these two acids precludes use of a starting solvent system with acetonitrile composition >67%. If only one of these two acids is present, however, a rapid analysis is feasible. A separation of this type is illustrated in Figure 2. The time required for this analysis is 70 minutes as compared with four hours for the separation shown in Figure 1. Finally, the presence of the phenacyl moiety as the exclusive chromophore at 254 nm permits direct quantitation
of molar ratios of these fatty acid derivatives based on peak areas. The relationship of peak area to moles of fatty acids remains linear over the range 100 ng to 100 pg.
CONCLUSIONS The method described above affords a rapid and convenient method for the derivatization and subsequent analysis of fatty acid mixtures on the microgram scale with a high degree of resolution in most cases. This method has been applied extensively to the analysis of fatty acids composition in chick fibroblast phospholipids (10) and of both free fatty acids and phospholipids in platelets (11). Modification of this procedure for the analysis of prostaglandins will be reported separately.
LITERATURE
CITED
(1) A. Grünert and K. H. Bassler, Fresenius Z. Anal. Chem., 267, 342 (2) (3) (4) (5)
(6)
(7) (8) (9) (10) (11)
(1973). U. Hintze, H. Róper, and G. Gercken, J. Chromatogr., 87, 481 (1973). H. Ehrsson, Acta Pharm. Sueclca, 8, 113 (1971). E. O. Umeh, J. Chromatogr., 56, 29 (1971). M. J. Cooper and M. W. Anders, J. Chromatogr. Sci., 13, 407-411 (1975). I. R. Plltzer, G. W. Griffin, B. J. Dowty, and J. L. Laseter, Anal. Lett., 6, 539 (1973). W. Morozowich, APhA Acad. Pharm. Sci. Abstr., 69, (19723). M. J. Cooper and M. W. Anders, Anal. Chem., 46, 1849 (1974). W. T. Moreland, Jr., J. Org. Chem., 21, 820 (1956). R. F. Borch and C. R. Moldow, unpublished results. R. F. Borch and G. H. R. Rao, unpublished results.
Received for review May
27, 1975. Accepted August 20,
1975.
Separation and Detection of Ortho-, Ryro-, and Tripolyphosphate by Anion Exchange Thin Layer Chromatography R. A.
Scott and G. P. Haight, Jr.
School of Chemical Sciences, University of Illinois, Urbana, Urbana,
III.
During work on the hydrolysis of various polyphosphates (in particular, pyrophosphate (PP¡) and tripolyphosphate (PPP,)) as catalyzed by an inorganic redox system (H2O2 oxidation of V02+) (1), a simple, rapid technique for the separation and identification of P¡ (orthophosphate), PP¡, and PPP¡
was
sought.
Much work has been done in recent years toward finding such a technique for the separation of polyphosphates during which various types of chromatographic separations have been examined including paper, (2-6) and anion exchange using thin layers (7-11). In many of these separations, the detection and visualization of the polyphosphates was accomplished by hydrolysis to P¡, followed by formation of some type of phosphate-molybdenum complex which is subsequently reduced to form the mixed oxidation state (Mo(V,VI)) polymer, phosphomolybdenum blue (PMB) (3-6). Problems have arisen due to catalysis of polyphosphate hydrolysis by acid eluents (2), slow hydrolysis of polyphosphate spots after separation (4-6), and visualization of the spot as PMB (4-6). Anion exchange chromatography can be carried out on thin layers by the use of cellulose impregnated with an appropriate anion exchanger coated on glass plates or plastic sheets. Berger et al. (10) used thin layers of Bio-Rex 5 ion
61801
exchanger to separate polyphosphates. Tanzer et al. (11) used polyethyleneimine (PEI)-impregnated cellulose-coated on glass plates and LiCl eluents to separate polyphosphates. For visualization, the chromatogram was sprayed with the reagent of Hanes and Isherwood (3), dried in an oven at 150 °C (to hydrolyze all polyphosphates), irradiated with uv light, and exposed to NH3 vapors to develop the blue spots. Iida and Yamabe (12) used NH4C1 and NaCl as the eluents and hydrolyzed the polyphosphates by spraying with an aqueous HNO3 solution before visualizing with molybdate and stannous chloride. The method presented here consists of the separation of polyphosphates (in particular, P¡, PP¡, and PPP¡) on PEIimpregnated cellulose-coated plastic sheets (thin layers) by use of LiCl eluents. The detection method used has the advantage of being simple and rapid, allowing visualization of polyphosphate spots in 5-10 minutes. This is accomplished by the simple expedient of using water to accelerate the hydrolysis process and PMB formation.
EXPERIMENTAL Thin Layers. Plastic sheets, 20 X 20 cm, coated with 0.1-mm cellulose (MN 300) impregnated with polyethyleneimine were obtained from Brinkmann Instruments, cut into 5 X 20 cm strips and stored at ~5 °C until used.
ANALYTICAL CHEMISTRY, VOL. 47, NO. 14, DECEMBER 1975
·
2439
