24. research methods · 2015. 4. 7. · 24. zinkel . research methods . 805 yearly instructions to...

24. Research Methods DUANE F. ZINKEL

USDA, Forest Service, Forest Products Laboratory, Madison, Wisconsin

GeneralAspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 804

Sampling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 805

General Separation Methods ........................ 805

Group Separations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 807

Isolation and Identification of Components by Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 809

Thin-layer Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . 810

ColumnChromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . 811

Argentation Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . 812

High Performance Liquid Chromatography . . . . . . . . 813

Gel Permeation Chromatography (GPC) and

Countercurrent Methods . . . . . . . . . . . . . . . . . . . . . . . . . 815

Gas-liquid Chromatography (GLC) . . . . . . . . . . . . . . . 816

Analytical Gas Chromatography ........................ 820

Qualitative Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 820

Fatty Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 820

Resin Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 821

Other Terpenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 835

Quantitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 835

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 837

HE UTILIZATION of any material, whether of natural or synthetic origin, depends, quite obviously, on the physical and chemical properties of

the material. To produce or further modify these materials and their properties in a reproducible fashion, reliable methods are needed to evaluate the properties and chemical composition. Such analytical methods define property parameters qualitatively (what) and quantitatively (how much). Factors such as speed, convenience, accuracy, equipment requirements, and size of sample are important considerations in the choice of available methods and procedures or the development of new pro

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cedures. Because the acquisition of quantitative analytical data usually represents a major portion of experimental efforts, a full understanding of the methodology is a prerequisite for successful completion of the research.

Analytical methodology can be categorized conveniently by end use, that is, for quality control or for research purposes. As in most such broad classifications, distinctions are not sharp. Thus, a quality control method can be used as a research procedure and vice versa. Often research methods become quality control methods as the methodology, usually instrumental, improves in dependability and convenience, and decreases in cost. An example of this is the gas chromatographic analysis of turpentine, which has largely replaced the earlier descriptive methods of distillation characteristics, specific gravity, and refractive index. A readily apparent difference between quality control and research analysis is seen in the qualifications of the person performing the analysis. Lundell in his 1933 paper (1), “The Chemical Analysis of Things as They Are,” divides those workers into two groups, the determinators and the analysts. The determinators (now usually called technicians) follow clearly delineated and standardized methods. In doing so, the technicians must be fastidious and have developed manipulative skills. The analysts, in addition to having a much higher degree of these skills, have developed through training and experience the ability to modify existing methods, to develop new methods, and to interpret the significance of analytical data. Thus, it is essential that all quality control service be under the supervision or surveillance of an analytical chemist.

In this section of the book, naval stores analyses also have been divided into quality control and research methods. The quality control methods for rosin and tall oil are presented in Chapter 26; the methods for turpentine are in Chapter 27. This chapter will encompass a range of research methods-from those that are simple but have not reached the regularity in use for formatting as formal standards to methods that are highly esoteric and require a high level of skill.

General Aspects

Both the analytical chemist and the technician must have an understanding of basic terminology and precepts in order to communicate with others. Just as with chemical compounds, standardized nomenclature is important in analytical chemistry. The terms analyze, identify, and determine are often misused. Simply, samples are analyzed, and specific components are identified or determined. Information on terms, spectroscopic nomenclature, and applicable SI units are found in the

24. ZINKEL Research Methods 805

yearly instructions to authors for Analytical Chemistry (e.g., Reference 2) and the “IUPAC Compendium of Analytical Nomenclature” (3). Yet, in the evolution of modern analytical methods, nomenclature also evolves. For example, it is now recommended that the mass/charge quotient for mass spectra be written m/z rather than the familiar m/e (where z is the standard symbol for charge number).

“Accuracy and Precision Revisited” is the astute title of an editorial by G. H. Morrison (4) about data acquisition in environmental chemistry. The concepts inherent in the title need to to be fully understood by any analyst in order to devise systems that provide valid results (a summary of validation is provided in Reference 5).It is not the intent of this chapter to review the basic tenets of analytical chemistry since they are discussed in most analytical chemistry texts. As a supplement to such texts, some other suggested appropriate publications are Guidelines for Data Acquisition and Data Quality Evaluation in Environmental Chemistry (6), Optimization of Chemical Procedures (7), Quality Assurance of Chemical Measurements (8 ), and How Good are Your Data Really (9). On the far end of the analytical spectrum is the limit of detection, a descriptive term for the lowest concentration of a component that is statistically different from an analytical blank (10). A true limit of detection is seldom of importance in most naval stores analyses with the exception, perhaps, of environmental samples.

Sampling. Sampling is a problem that must be addressed in all analytical work (11-15).When the limiting factor in analytical uncertainty is in the sampling, overall results may be improved by using a rapid method of low precision that permits the analysis of more samples, thus giving an average value of greater confidence. In some involved analytical schemes, however, special efforts are needed to devise methods that provide representative samples. Kraft pulping black liquors, tall oil soaps, and wood are particularly difficult to sample. The black liquors, tall oil, or tall oil soaps can be non-homogeneous owing to stratification, whereas wood in the tree or even as chips can have a high degree of variability owing to intra- and intertree differences as well as differences between species. Attempts a t solving sampling problems are given in the literature for black liquor (16,17) and for maintaining black liquor samples (18), tall oil (Chapter 26), and tall oil soaps (19); sampling problems of wood was a major consideration in lightwood studies (summarized in 20,21), particularly for large numbers of trees (22).

General Separation Methods. The classical methods for separation and analysis of the “extraneous components’’of wood as used in the mid-

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1950s to 1960s have been published (23,24). While some of the basic methodology continues to be used, dramatic changes have occurred in the intervening years.

With the exception of products from process streams such as turpentine, rosin, and distilled tall oil, the precursor naval stores materials are present in the wood matrix from which they must first be removed for analysis. The most common method is the continuous Soxhlet extraction with an organic solvent. However, the nature and specifics of application of the solvent determine the quantity and types of materials extracted. Solvents such as acetone or ethanol-benzene [now usually replaced by ethanol-toluene (25) because of the carcinogenicity of benzene] will extract significant amounts of materials other than oleoresins and fats. On the other hand, hydrocarbon and aromatic hydrocarbon solvents tend not completely to extract these materials in that the rate of solvent permeability into the wood coupled with water immiscibility affect the extraction rate. The boiling point of the solvent establishes the pot temperature of the Soxhlet extraction, which influences the stability of the extractive components. A solvent must not react with the extractives (esterification by alcohol solvents is a potential problem). Predrying, particularly ovendrying, of wood before extraction decreases the quantity (and alters the relative composition) of extractable materials. For example, Nelson et a1 (26) have shown ovendrying will decrease even the petroleum ether-soluble materials by a factor of almost two. Thus, it is only logical to maintain a wood sample in the “green” condition for extraction. For many research studies, diethyl ether is the best solvent. Extraction of Wiley Mill ground wood for 8 hours is usually adequate, but the extraction period should be increased to 24 hours or more for critical material balance experiments. Methyl tert-butyl ether (b.p. 55°C) with its low susceptibility to peroxide formation may be of value in special situations. In lightwood studies, xylene percolation was the most widely used extraction method (27). An important feature of this extraction method is the codetermination of water content. Although this extraction method gives higher values for turpentine than the standard caustic cooking method (28) with which it was compared, its efficacy with respect to effective solvents such as diethyl ether has not been established. Supercritical solvent extraction (CO2) has been investigated as a processing method for naval stores recovery (Chapter 5), but there are no reports in the literature on use of the technique for extraction in analytical situations. However, supercritical fluid chromatography/gas chromatography/mass spectrometry (SFC/GC/MS) has been applied to the essential oils in spruce needles and cedar wood (29). The only commercially available apparatus for CO2 extraction (J&W High Pressure Soxhlet Extractor, J&W Scien-

24. Z I N K E L Research Methods 807

tific Inc., Rancho Cordova, CA) has a very limited sample capacity. In the laboratory, turpentine is usually recovered from wood by steam

distillation from caustic solution (28). To avoid the problem of bumping during distillation, ground wood (Wiley mill) is tied in a cheesecloth bag, and the bag is placed on a supporting metal screen in the resin kettle. The total volume of distilled turpentine is measured directly. Condensor outlets should be properly vented and exposure to sample vapors should be minimized through the use of well-ventilated rooms to avoid a potentially toxic material present in such distilled turpentines (30). The composition of the turpentine is then determined by gas-liquid chromatography (GLC) (Chapter 27). A wood extract can also be used directly for turpentine determination (both quantity and composition) such as was done for the high n-pentane turpentines of Jeffrey pine (31). Other methods of more specialized applications are the isolation of turpentine by extraction from an ethanolic solution of KOH neutralized oleoresin (32) and the “closed loop stripping” of volatile oils from conifer needles (33,34), which are similar to head space analysis (35; as applied to monoterpenes from Pinus sylvestris needles, 36, and other pines, 37). Cryotrapping as used for determining monoterpenes in forest air (299), could be applied to analysis of ambient air in wood and naval stores processing plants.

The expression of analytical data is usually given as a portion of sample on a weight basis, and sometimes on a weight/volume basis. For data correlations and comparisons among various laboratories, such basis must be clearly defined. Otherwise, great confusion and uncertainty are the result, such as the problem that plagued the Pulp Chemicals Association for many years in assembling tall oil and turpentine recovery data because of varying and incorrect definitions for a cord of wood. For any scientific discussion and comparison, the most constant and common denominator on which to express extractives (and turpentine) yield data is the wood substance, that is, the ovendry extractive-free (ODEF) material. This approach has been particularly important for situations involving high contents of extractives such as with heartwood or with induced lightwood.

Group Separations. Obtaining general information on component types is the first step in most research and quality control analysis. Isolation procedures in the laboratory and the separation process in commercial operations often have a number of elements in common as they are derived from common chemical principles, such as the volatility of the essential oil (i.e., turpentine), or the relative volatility of monomeric as opposed to polymeric fatty and resin acids (determinated in the laboratory by a microdistillation, 38). Thus, “volatile” material is separated from

808 NAVAL STORES

nonvolatile components by distillation. However, the nonvolatile portion in the analytical laboratory, particularly when studying naval stores precursors as they are present in the wood, is often prepared by an independent method to avoid transformations of the components during distillation.

The nonvolatile portion of naval stores materials consists of acidic and neutral components. The acid fraction (the content is characterized by acid number, see Chapter 26) can be separated from the neutrals fraction by several methods. The classical method of separation involves partition of the neutral components into a water-immiscible solvent and of the acids, usually as the sodium salts, into an aqueous phase. Although the classical method is satisfactory for preparative use, it does not provide a quantitative separation because of problems associated with the characteristics of soap (sodium salts of water-insoluble fatty and resin acids) solutions. However, quantitative separation of neutral from acidic materials can be accomplished using DEAE-Sephadex ion exchange (39,40). The acidic materials are exchanged onto the diethylaminoethyl groups, which are the base form, while the neutrals pass through the column. Elution with carbon dioxide saturated solvent provides a gentle yet quantitative procedure for recovering the acids. The abietadienoic acids, particularly levopimaric acid, are not isomerized. Three magnitudes of separation have been developed: (1) ca 100-200 mg macro scale for gravimetric determination of acids and neutrals (40) and giving sufficient neutrals for saponification (particularly useful with wood extractives), (2) ca 10 mg semimicro scale (41) for isolation of acids for GLC when removal of neutrals is desired but determination of neutrals content is not needed, and (3) sub-milligram scale (42) designed for extractives of oleoresin from portions of plant, material such as sections of growth rings or individual pine needles. If a simple flame ionization system were available as a universal detector, the neutrals in the semimicro and micro methods could be estimated. For a more complete discussion of neutrals and nonsaponifiables, see Chapter 11.

Total neutrals content can be determined by quantitatively removing the acidic materials such as by absorption on an alumina column. One such method has used basic alumina to remove acids from an acetone solution (43,44); correction factors have been reported to convert data from a neutrals content basis to a nonsaponifiable basis. Separation of neutrals by compound type is usually accomplished by a series of absorption chromatographic methods but often with specific precipitation or derivative methods as part of the sequence, such as in the definitive publication of Conner and Rowe (45; see also Chapter 11).

Although most naval stores analyses involve neat materials from pro-


cessing streams or solutions of extractives, the dilute nature of the aqueous samples from pulp mill effluents or bodies of water often requires novel approaches in isolating fatty and resin acids for analysis and determination of biological activity. One widely used approach has been to extract the acids and neutrals onto Amberlite XAD styrene-divinyl benzene polymers (46,47). This methodology and alternative solvent extraction methods have been reviewed (48-50).

The class separation of resin acids from fatty acids can use two principle differences: (1)steric hinderance differences a t the carboxylic acid and (2) size differences between the more rigid resin acids and the, acyclic, fatty acids. Using steric hinderance of the carboxylic group, the fatty acids can be preferentially esterified, such as used in quality control analysis (Chapter 26), and the resin acids separated from the fatty acid esters as discussed above. Resin acids can be separated from fatty acids by thin-layer chromatography or column chromatography on silica gel with solvents such as 1:1 petroleum ether:diethyl ether. Fatty acids can be separated from resin acids as the methyl esters on the basis of molecular size by gel-permeation chromatography (GPC) using 40-60 Å porosity Styragel packing and diethyl ether as solvent (51). While the separation of fatty acids from tricyclic resin acids is effective with this system, the presence of bicyclic labdane resin acids (particularly found in pine needles) can cause complications as the labdane methyl esters elute between the fatty acid esters and the tricyclic resin acid esters. In the older literature, cyclohexylamine precipitation (crystallization) was used frequently to separate the resin acids from solutions of oleoresin, extractives, or tall oil (an example is given in Reference 52). The recovery of resin acids from oleoresins by this method is said to be quantitative. Specific amines are selective in the isolation of pure resin acids by crystallization of the amine salts (Chapter 9).

Oxidation artifacts can usually be removed from methylated resin acid and/or fatty acid fractions by absorption chromatography. Silica gel is generally more useful when it is desirable to recover and further separate the oxidized components. For simple removal of oxidized components, small samples of methyl esters in a low-boiling hydrocarbon such as pentane or hexane can be passed through a short (2-3 cm) bed of neutral alumina activity III in a Pasteur pipette.

Isolation and Identification of Components by Chromatography

Physical separation and isolation of a component is a precursor to identification. Modern chromatographic methods, however, permit identification that is a t least tentative based on reproducible chromato-

810 NAVAL STORES

graphic behavior. This is particularly true for gas chromatography. The separation and identification is the qualitative aspect of chromatography. In this section of this chapter, qualitative and preparative chromatographic methods are reviewed that are of current application or are of potential use in naval stores research.

The last three decades have seen an explosive growth in chromatography. The pertinent original literature on chromatographic separation of the types of components in naval stores materials or precursors, i.e., the related terpenes, fatty acids, etc. from all plant sources, is too immense to review in-depth in this chapter. An overview on terpenes can be found in the Journal of Chromatography Library (53,54). In regard to the volatile components of naval stores, turpentine quality control analysis is treated in Chapter 27. The primary thrust of the remainder of the present chapter will be with respect to the nonvolatile components.

Thin-layer Chromatography (TLC). The use of a thin layer of chromatographic media on a glass plate, an aluminum sheet, or a plastic film is a more recent version of column chromatography. The technique is simple in equipment needs and is rapid. Sometimes, the separated components can be seen as “spots” by visualization with UV light, but more often specific reagents are sprayed onto the plate. Some approaches used in TLC over the years are given in a publication on wood pulp extraction (55). Methodology for densitometric quantitation of wood extractives has been described by Pensar (56 ) . Important facets of TLC in lipid separations can be found in the reviews of Mangold (57) and Privett et al (58 ) . The various absorption systems have the most utility in class separations of lipids.

TLC has been used with monoterpenes (59), but applications are somewhat limited by the volatility of the solutes. Applications of TLC to diterpenes includes several publications on rosin (60-63 ) and modified rosins (64-66 ). TLC has been used to separate acids and neutrals by application of ethanolic sodium hydroxide a t the origin followed by application of the sample (67). The DEAE-Sephadex method, however, is more convenient and quantitative. In another application, TLC was used to separate reaction products of resin acids with methylene chloride; ester products were recovered from the plates and amounts determined by ultraviolet spectroscopy (68) . Because of difficulties in quantitative recovery of components after separation, TLC is most often used as a rapid method for devising solvent systems for column chromatography or as a small scale preparative method using thick-layer plates.

Perhaps the most significant contribution of TLC has been in the development of argentation chromatography. In addition to numerous


reports for fatty acids (e.g., 69-71), argentation TLC has been widely used as typified by publications in the 1960s on monoterpenes and sesquiterpenes (72-74) and on triterpenes (75-78). Tl (79) and Cu(I) (complexes are of potentially greater stability than Ag+, (80)) and can be used, but it is difficult to maintain Cu in the reduced (+1) state; thallium salts are highly toxic. Although chromatography of terpenes has been reported with silver ion in the mobile phase (fiberglass papers impregnated with hexadecane) for sesquiterpenes (81) and resin acid esters (82), incorporating the silver in the stationary phase is less cumbersome and less costly. In general, the negative counter ion of the silver salt is not of significance to chromatographic efficiency, but one report notes advantages of silver iodate over silver nitrate in TLC of certain oxygenated terpenoids (83). Argentation TLC of resin acid methyl esters has been described (84,85). A representative chromatogram is shown in Figure 1 for AgNO3-alumina with diethyl ether/petroleum ether solvent (84); AgNO3 on silica with benzene as eluting solvent (85) gave some differences in elution order. Promising argentation TLC of dihydro-resin acid esters (D.F. Zinkel, unpublished, Figure 1a) led to to successful preparative column chromatographic preparation for spectra purposes (86).

Column Chromatography. Obviously, column chromatography parallels TLC to a great extent, but some chromatographic operations are not amenable to TLC. A recent book by Hostettmann et al (87) provides a good introduction to the application of preparative absorption and partition chromatography in natural products isolation. Absorption chromatography is most useful in separating components based on type and amount of oxygenation. Examples have been given in the above discussions on group separations and TLC: silica and alumina absorption chromatography have been used to separate and isolate oxygenated resin acids from pine needles (88). Other examples of column chromatography applied to terpenes are found in a short review (54).

Reverse phase column chromatography for saturated fatty acids, developed by Ramsey and Patterson in 1948 (89), has been used to prepare highly purified fatty acids (90). The method was applied to resin acids, for example, in the isolation of palustric acid (91), the separation of pimaric and isopimaric acids (92), the analysis of resin acid composition (93), the study of thermal isomerization of levopimaric acid (94), and the isolation of Diels-Alder adducts of levopimaric acid (95). Although the reverse phase methodology was invaluable for a short period of time, it was soon supplanted by GLC for analytical purposes and by other chromatographic methods, such as argentation chromatography, for preparative purposes.

NAVAL STORES812

Figure 1. Argentation thin-layer chromatogram (40% w/w AgNO3/neutral alumina) of fa t ty and resin acid methyl esters. a. Methyl: ( 1 ) isopimarate, (2) leuopimarate, (3) palustrate, (4) pimarate, (5) abietate, (6) neoabietate, (7) dehydroabietate, (8) dihydro and tetrahydro resin acids, (9) linoleate, (10) oleate, and (11) stearate. ( A ) Resin acid methyl ester mixture prepared f r o m pure resin acids, (B) resin acid methyl esters from oleoresin, (C) resin acid methyl esters from g u m rosin, and ( D ) fat ty ac id methyl esters. Developed with 25% Et 2O/ petroleum ether (84). b. Hydrogenated resin acid methyl esters. Methyl (11) 8(14)-pimarenoate. (12) 13-abietenoate, (13)8-abietenoate, (14) 8(14)-abietenoate, (15) a n abietanoate, (16) 7-isopimarenoate, (17) a n isopimaranoate, (18) dehydroabietate, (19) 8(14)-isopimarenoate, and (20)8-pimarenoate. Developed with petroleum ether (D. F. Zinkel, unpublished, 1966).

Argentation Chromatography. Argentation column chromatography has been widely applied to fatty acid isolation and purification since the early reports of de Vries (96,97) and Privett and Nickell (90). Argentation chromatography using either silica or alumina supports has been used to isolate resin and fatty acid methyl esters from distilled tall oil (98), methyl strobate (99), anticopalate (100) , imbricataloate (101), 8,15isopimaradien-18-oate and other diterpenes from Pinus quadrifolia (102), a number of new resin acid esters from pine foliage (88,103,104), dihydroabietates (105), neutral diterpenes from Jack pine bark (106), and east-


ern white pine cortex oleoresin (107), and to prepare high purity resin acid methyl esters for standard spectra (86).

Argentation chromatography with the silver fixed in the stationary phase as the salt of a cation exchange resin is an obvious extension of the general methodology. Although such a chromatographic system was reported for fatty acid methyl esters as early as 1963 (108), the limited ion exchange capacity of the resins severely restricted efficacy of the methodology. However, this problem was alleviated later by the availability of the Amberlyst XN-1010 resins with superior ion exchange capacity and, hence, separation capability for fatty acid esters (109). This argentation resin chromatography has been adapted to the resin acids, both for the methyl esters and the free acids using ether/petroleum ether and acetone as eluents (110). Other advantages of argentation resin chromatography over the analogous Ag+/silica or alumina systems are that the resin columns can be reused over periods of years (we have used a column for over five years) and the loss of esters through hydrolysis of fatty acid esters (111) and resin acid esters (110) is avoided. The primary disadvantages of the system are the tedious preparation of silver-resin and some remaining limitations on column efficiency. Argentation resin chromatography has also been successfully applied to purification of resin acid dimers (112).

High Performance Liquid Chromatography (HBLC). HPLC has become a highly popular and widely used varient of column chromatography (113), with a wide variety of equipment and accessories available commercially. Although H P is officially designated “high performance,” both “high pressure” and “high performance” were used for several years; the small particle size in the columns require higher pressures than usual column chromatography. A recent editorial suggests the removal of HP to give the generic LC, noting that there is no deliberate low performance chromatography (114). HPLC with silica columns has been used to analyze volatile terpenes (115,116), menthols (117), monoterpene acids as their naphthylethylamides (128), labile sesquiterpenes a t low temperature (119), and triterpenoids (120). We have resolved two isomers of methyl 9,10-secodehydroabietate on an analytical silica column (eluting solvent 0.5% methyl-tert-butyl ether/heptane) but were unsuccessful in obtaining the resolution on a preparative scale. The separation, however, has been more successful with ß-cyclodextrin columns. (D.F. Zinkel, unpublished). There have been numerous publications on the application of HPLC using reversed phase packings to fatty acids such as the determination of free fatty acids in oils and alkyl resins (121), but much of the effort has involved derivatives such as napthacyl

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(122), phenacyl (123-125 ), and coumarin (126-127 ) esters to improve analysis with ultraviolet and fluorescence detection. Reverse-phase HPLC of glycerides has been reviewed and extended (128). A practical guide reviewing the HPLC of lipids has been published (301). It has also been applied to quantitative determination of the major lipid classes (flame ionization detector, 129), fatty acid dimers (130), monoterpenes (131 ), essential oils (132 ), triterpenes (120,133), and steroids (134,135). More specific to naval stores is the application of HPLC to the analysis of dehydroabietic acid in kraft mill effluents (136-138 ), the analysis of modified and unmodified resin acids (139) and tall oil (140,305), the determination of rosin in shellac (141), and the isolation and analysis of the chlorodehydroabietic acids (142) and their biodegradation products (143 and references therein) as the imidazoles. Argentation resin HPLC has been adapted to fatty acid esters (144-147).

The step from chromatography as an identification tool to a quantitative tool is a major step fraught with many difficulties. The key to good quantitative analysis is a suitable detector. Most liquid chromatography (LC) detectors are of a flow-through type and can be divided into two groups (148): those that measure bulk properties of the eluate, such as refractive index detectors, and those that selectively monitor specific characteristics of the solutes. The infrared detector (carbonyl region) has been used for fatty acids (149) and triglycerides (150), and it would appear to be a valuable detector for HPLC analysis of naval stores. However, such use is greatly limited by compatability of solvent with the materials available for cell windows. Attempts have only recently been successful in a practical sense in developing a flame ionization detector as a universal LC detector.

Based on the published literature and our unpublished experiences at the Forest Products Laboratory with resin acids, fatty acids, their methyl esters, and some other related compounds (using several partition columns and various solvent systems), HPLC offers little potential for novel approaches to difficult analytical problems that cannot be solved by analytical procedures based on GLC. However, the special features of supercritical fluid chromatography may provide useful separations, and the method has the advantage of simplicity in solvent removal. Although some preliminary data have been published on the separation of abietic and neoabietic acids (151), free fatty acids (infrared detection, 152 and flame ionization detection, 153,154), glycerides (155), and fatty acid dimers (156), more rapid application of this facet of HPLC has been constrained by limited commercial availability of equipment-asof this writing, specific supercritical fluid HPLC instrumentation has recently been introduced by two manufacturers.


From a preparative view, however, HPLC is useful for separating fatty acid methyl esters (157), retenoids (158), a peak skimming-recycle technique applied to unsaturated labdane isomers (159), and to isolating labdane acids from podocarp oleoresin (160), and abietic-type acids from Pinus ponderosa (300). The broad area of preparative liquid chromatography (LC, TLC, and GPC) is covered in a 1987 volume of the Journal of Chromatography Library Series (161).

Gel-permeation Chromatography (GPC) and Countercurrent Methods. GPC provides a chromatographic method that operates by a different mechanism, that of molecular size. Most of the GPC separations in the literature relate to polymeric materials, but there are important applications for low molecular weight materials. A number of separations of fatty acids or derivatives by means of Sephadex materials have been reported (162-166); the most significant applications used polystyrene (cross linked with divinylbenzene) gels. Pertinent applications of GPC include monoterpenes (167,168), fatty acids (169), pyrolytic tars (including wood-derived tars, 170), coatings (171) and alkyds (172), polymerized fatty acids (173-175), polyamides (176), deposits in pulp and paper production (177-178), and the determination of rosin in shellac (141). A GPC method has been developed to separate fatty acids from resin acids as methyl esters (Table 1)to simplify subsequent GLC analysis of the com-

Table 1. Representative GPC Elution Volumes of Resin and Fatty Acid Methyl Esters (51).1

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ponents (51). This method has been used in prefractionation in the isolation and identification of new resin acids; the method was the key in the identification of anticopalic acid (100) in the bark and wood of western white pine (methyl anticopalate and methyl isopimarate have nearly identical GLC retention characteristics for polar and nonpolar packed columns, a situation that had previously lead to misidentification).

A number of laboratories are using the GPC method of Chang (179)for internal quality control of rosin dimerization. The potential for GPC in the characterization of naval stores and products can be seen in the chromatographic illustrations of Figure 2, taken from the report of Bentejac (180). GPC of rosin and rosin glycerides is also depicted in commercial literature (181). In considering the extension of the above GPC applications to other materials, attention should be given to the fundamental principles of GPC separations (182).

One of the more recent developments among liquid chromatographic methods is high speed countercurrent chromatography (CCC) (183,184), a group of liquid-liquid partition techniques consisting of several varients such as droplet CCC, rotation locular CCC, and coil planchet centrifugation. These techniques are based on Craig countercurrent extraction but are more rapid and efficient. Although most natural product applications (185-188) to date have been with compounds more polar than the usual lipophilic materials of naval stores, other applications include fatty acids (189,190) and essential oils (191), the latter with compressed CO2 as mobile phase. CCC can be expected to become an important adjunct in laboratory separations and purifications of naval stores components.

Gas-liquid Chromatography (GLC). GLC has become the mainstay in the identification and determination of the components in naval stores materials. Details on the analysis of turpentines and other neutrals are given in chapters 27 and 11,respectively; identification of resin and fatty acids by GLC is discussed in detail on pages 820-837 of this chapter. Preparative GLC is a useful but not extensively used method for isolation and purification. Although production-scale GLC units are commercially used for preparation of high-value fragrance components (Chapter 12), most preparative work in the research laboratory is done with analytical packed columns with repetitive injection and component collection. Many commercial laboratory preparative units have been poorly designed in that the abrupt cooling of the effluent, rather than a gradient cooling for efficient condensation (lengths of stainless steel hypodermic tubing are more effective than glass tubing), results in aerosols which are difficult to condense or trap. We have made effective use of preparative GLC for purifying resin acid methyl esters, particularly the methyl tetrahy-


Figure 2. Gel permeation chromatography of naval stores materials and derivatives (system of four µ-Styragel columns, 1000 Å, 500 Å, 100 Å, and 100 Å; Tetrahydrofuran as solvent and a differential refractometer detector. Point E is 23 ml f r o m injection. All components eluted within 20 ml of E.). F=fat ty acids, R=resin acids, M = monoterpenes, g=glycerine, p=pentaerythritol, FP= fumaropimaric acid, MP=maleopimaric acid, gFx= various (x=1,2, or 3) fa t ty acid glycerides, gRx=various resin acid glycerides, pRx= various pentaerythritol esters of rosin, FF=fatty acid dimers, RR=rosin dimers, and g(RR) x and p (RR) x are the glycerol or pentaerythritol esters of dimerized rosin (180).

818 NAVAL STORES

Figure 2 (continued). Gel permeation chromatography of naval stores materials and derivatives (system of four µ-Styragel columns, 1000Å, 500Å, 100Å, and 100 A; Tetrahydrofuran as solvent and a differential refractometer detector. Point E is 23 ml from injection. All components eluted within 20 ml of E.). F=fatty acids, R=resin acids, M=monoterpenes, g=glycerine, p=pentaerythritol, FP=fumaropimaric acid, MP=maleopimaric acid, gFx= various (x=1,2, or 3) fatty acid glycerides, gRx= various resin acid glycerides, pRx=various pentaerythritol esters of rosin, FF=fatty acid dimers, RR=rosin dimers, and g(RR)x and p(RR)x are the glycerol or pentaerythritol esters of dimerized rosin (180).


Figure 2 (continued). Gel permeation chromatography of naval stores materials and derivatives (system offour µ-Styragel columns, 1000 Å, 500 Å, 100 Å, and 100 A; Tetrahydrofuran as solvent and a differential refractometer detector. Point E is 23 ml from injection. All components eluted within 20 ml of E.). F=fat ty acids, R=resin acids, M=monoterpenes, g=glycerine, p=pentaerythritol, FP=fumaropimaric acid, M P = malenpimaric acid, gFx = various (x = 1,2, or 3) fa t t y acid glycerides, gRx=various resin acid glycerides, pRx= various pentaerythritol esters of rosin, FF=fat ty acid dimers, RR=rosin dimers, and g(RR)x and p (RR) x are the glycerol or pentaerythritol esters of dimerized rosin (180).

820 NAVAL STORES

dropimarates and tetrahydroisopimarates (86,192). One must be alert, however, to transformations that can occur during GLC, such as the facile dehydration of 15-hydroxydehydroabietate on packed columns to form the abietatetraenoate (193). Preparative gas chromatography is the subject of a 1971 book (194) that contains applications to turpentine, essential oils, and lipids (also see 195).

Specifics on the chemistry and characterization of naval stores components are covered in Chapters 9-11. Usually, components are isolated by chromatographic methods and then characterized by combinations of infrared, ultraviolet, nuclear magnetic resonance, and mass spectrometry. The tandem combination of chromatographic separation and spectrometric analysis, i.e., the “hyphenated methods,” can be useful in the rapidity of data acquisition, but the information developed is generally limited to comparative identification of known compounds. Such tandem equipment is relatively expensive a t this time and often limited in availability. The most common of the hyphenated methods is gas chromatograph-mass spectrometry (GC-MS), providing GLC retention characteristics and mass spectra patterns for tentative identifications.

Analytical Gas Chromatography

Qualitative Aspects. The application of GLC as a tool in the isolation and characterization of naval stores components has been reviewed in the previous paragraphs. The primary use of GLC, however, is analytical. This includes the identification of the constituents of fatty acids, resin acids, and other terpenes in extractives and naval stores materials as well as their quantitative determination.

FATTY ACIDS. One of the earliest and most intensive applications of gas chromatography was in the area of fatty acids. Because the subject has been adequately reviewed for both packed (e.g., 196,197) and open tubular or capillary (e.g., 196-198) columns, only some of the pertinent facets will be summarized here.

Although it is often desirable to determine the composition of fatty acid samples in the free acid form, the results using methyl esters are far more satisfactory. Preparation of the methyl esters from the free acids can be accomplished quantitatively by a number of methods, the most common of which are esterification with methanol using boron trifluoride (199; BF3-ether, 200) or HCl as catalyst, pyrolysis of the tetramethyl ammonium salt in the injection port of the gas chromatograph, or reaction with diazomethane. The conditions for quantitative methylation of fatty acids with diazomethane were studied by Schlenk and Gellerman (201);


optimum methylation is achieved in diethyl ether-methanol. Fatty acids present as triglycerides, such as in wood extractives, can be transesterified to the methyl esters using methoxide-catalyzed methanolysis (202), a method that can be accomplished using robotics (203). The primary fatty acids of tall oil are the unsaturated C-18 oleic and linoleic acids. Smaller amounts of other fatty acids are also present, including other C-18 and C-20 unsaturated acids and some saturated acids; a complete treatment can be found in Chapter 10.

Although nonpolar columns are useful in the analysis of fatty acid esters, such as in the analysis of metal soaps in brownstock pitch deposits (204), polar columns are usually necessary and preferable to obtain resolution of the many unsaturated fatty acid components. Indeed, most of the early gas chromatographic analysis of tall oil fatty acids used polyester coated column packings such as ethylene glycol succinate (EGS, 205-207), ethylene glycol succinate-silicone copolymer (EGSS-X, 206,207) and diethylene glycol succinate (DEGS, 208-210). The efficacy of capillary columns with butanediol succinate (BDS) coating has been applied to fatty acids in tall oil (211 ). In this work, the temperature dependency of retention was used to aid in distinguishing the degree of unsaturation of unknown components. The cyanosilicone liquid phases may be useful in resolving unsaturated geometric isomers but may have limitations with respect to positional isomers (212). (A recently described OH-terminated cyanosilicone gives retention ratios greater than 1.0 for methyl linolenate/arachidonate (18:3/20:0)(213), thus, indicating a potential for greater selectivity over other cyanosilicones for other unsaturated fatty acids.) These liquid phases also have much lower sample capacity than do the polyesters with respect to overloading, but, on the other hand, they are preferred in GC-MS because of lower liquid phase bleeding (214).

The most extensive set of retention information for tall oil fatty acid methyl esters is given in the capillary BDS paper (211); one should note, however, that many of the fatty acid identifications, though based reasonably on MS and GLC retention data, are but tentative. Thus, there is no single, definitive publication on the retention characteristics of tall oil fatty acids. Research in this regard is in progress in the author’s laboratory.

RESIN ACIDS. The first application of gas chromatography to resin acid (methyl ester) analysis was reported in 1959 by Hudy, who used a packed column containing the polyester BDS (215 ). Subsequently, a number of publications described the GLC characteristics of resin acid esters for packed columns with a variety of liquid phases; these publications have

822 NAVAL STORES

been reviewed (41,86). Reference 41 also reviews the state of the art of resin acid GLC, which includes capillary columns.

As with the fatty acids, gas chromatography of resin acid esters (usually the methyl esters) is far more effective than for the free acids. Diazo-methane is the most effective reagent for preparing the methyl esters. The reaction is instantaneous and quantitative in 9: 1diethyl ether:methanol. However, many laboratories are very concerned about the hazards of using diazomethane, particularly in regard to technicians in quality control laboratories. Thus, other methods of alkylation have been investigated. Resin acid methyl esters can be prepared with the analogous trimethylsilyldiazomethane, but the specifics for quantitative conversion have not been defined. Trimethylsilyldiazomethane is reported to be a safe, stable, conveniently handled liquid, and it is commercially available as a 10% solution in heptane. The cost of the reagent is considerably higher than that of diazomethane. Another popular methylation method for resin acids involves the preparation of the methyl esters by pyrolysis of the tetramethylammonium (TMAH) salts in the injection port of the gas chromatograph (216). It is recommended that the acids (either resin acids, fatty acids, or tall oil acids) be titrated with TMAH/methanol to a pH of 12.0 to 12.5 with a pH meter rather than to the pink endpoint using phenolphthalein (217).

Other esters of resin acids (prepared by diazoalkylation or by reaction with dimethylformamide dialkyacetals) have been evaluated for potential improvements in the GLC separation characteristics of the resin acids in rosins (218). The ethyl ester has been useful for differentiating between co-occurring pinifolic acid (a dicarboxylic labdane) and its monomethyl ester in Pinus nigra foliage (88). Interestingly, the esterification with diazoethane does not require the presence of methanol for rapid reaction. Ethyl esters prepared by reaction with triethyloxonium tetrafluoroborate are being used in the analysis of resin and fatty acids in pulp mill effluents (50).

Polar liquid phases are more effective than nonpolar liquid phases in the gas chromatographic separation of methylated diterpene resin acids of rosin. This is because of selectivity between the polar sites of the liquid phase and the unsaturation (double bonds) in the resin acids. Although we used DEGS packed columns in the early years of our research, EGSS-X (cf low-loaded columns, 206,207) has since been the liquid phase of choice for packed columns because it is more stable, yet it has almost identical characteristics to DEGS for resin acid methyl esters (41,219). The cyanosilicones have polarity characteristics similiar to the polyesters and could be the liquid phases of choice because of their greater stability. Indeed, the resolution of levopimarate and palustrate, previously not at-


tained with polyester packings, was achieved with a cyanosilicone liquid phase but with loss in resolution of the important dehydroabietate/ neoabietate pair (218). However, the major drawback that we and others have experienced with the cyanosilicone packings is the difficulty in preparing and maintaining satisfactory columns.

Attempts have been made to improve column selectivity by preparing various alkyl esters of resin acids (using the dimethylformamide dialkylacetals, noted above), particularly to prepare more hindered esters. With the possible exception of the tert-butyl ester for special purposes, most of the other alkyl esters did not provide any advantages over the methyl ester. The hindered trimethylsilyl esters can be prepared, but they are extremely labile to hydrolysis, and, even if handling problems can be avoided, these esters do not survive reaction with the polyester column packings (220). For the packed column gas chromatographic analysis of resin acids in rosin, it is recommended, therefore, that the methyl esters be chromatographed using a column packed with 10% EGSS-X (41,219). Representative chromatograms are shown in Figure 3.

Although packed columns are in widespread use in quality control laboratories for analyzing resin acids in rosin, most research laboratories have opted for the high efficiency of capillary columns. (Jennings (221) has published a succinct review of important facets in capillary GLC). Capillary column retention data for resin acid methyl esters have been reported for a number of liquid phases: SE-30 and other methyl silicones (222-226), SE-54 (223), Apiezon L (226), PEG(223,225,226), FFAP(224), EGS/DEGS (227), DEGS(224), cyanosilicones (223), and BDS (222224,226,228). BDS, the first liquid phase reported for GLC of resin acids, is the most useful of the liquid phases in capillary GLC for the common resin acids or rosins, resolving all of them including the levopimarate/ palustrate pair.

For most analyses of the resin acids in rosins, a BDS column provides sufficient efficiency for resolution along with a short analysis time (Figure 4); column efficiency is about 2300 plates/meter with methyl neoabietate eluting under 15 minutes a t 180°C (228). Details on the effect of column temperature on retention and separation factors have been published (228 ). The primary limitation of polyester liquid phases such as BDS is the long retention of oxygenated derivatives such as imbricataloate, acetylisocupressate, acetylimbricatoloates, and the corresponding deacetylated derivatives (the retentions of the latter alcohol-type derivatives are so long as to be effectively lost on the column) that are found in slash pine oleoresin and rosin (229) and ponderosa pine oleoresin (230). When such oxygenated resin acids are present (this is particularly the case with oleoresins from pine foliage), the use of a nonpolar stationary

824 NAVAL STORES

Figure 3. Gas chromatograms of typical tall oil, wood, and gum rosins (new 10% EGSS-X column; 8 ft. × 1/8 in., 200°C). 1 =pimarate, 2=sandaracopimarate, 3=communate, 4/5=leuopimarate/palustrate, 6=isopimarate, 7=abietate, 8= dehydroabietate, and 9=neoabietate (41).


phase reduces analysis time to within reasonable limits. For example, a methyl silicone such as the familiar SE-30 or the bonded DB-1 column will resolve all the common resin acids if the column temperature is about 165°C or lower. Generally, this analysis can be completed isothermally in 20 minutes (302) but may require a temperature program if higher molecular weight components are present in the oleoresin such as suc-

Figure 4. Gas chromatograms of resin acid methyl esters from slash pine oleoresin and rosin using a 7-m BDS fused silica column, 180°C. 1 =pimarate, 2=sandaracopimarate, 3=communa te , 4=leuopimarate, 5=palustrate, 6=isopimarate, 7=abietate, 8=dehydroabietate, and 9=neoabietate (41).

Tab

le 2

. R

eten

tion

Cha

ract

eris

tics

of

Dit

erpe

ne R

esin

Aci

d M

ethy

l E

ster

s on

Cap

illar

y G

LC

Col

umns

. R

eten

tion

s Rel

ativ

e to

Met

hyl P

imar

ate.

Tab

le 2

(co

ntin

ued)

. Ret

enti

on C

hara

cter

isti

cs o

f Dit

erpe

ne R

esin

Aci

d M

ethy

l Est

ers

on C

apill

ary

GLC

Col

umns

. Ret

enti

ons

Rel

ativ

e to

Met

hyl P

imar

ate.

Tab

le 2

(con

tinue

d). R

eten

tion

Cha

ract

eris

tics

of D

iter

pene

Res

in A

cid

Met

hyl E

ster

s on

Cap

illar

y G

LC C

olum

ns. R

eten

tion

s R

elat

ive

to M

ethy

l Pim

arat

e.

Tab

le 2

(con

tinue

d). R

eten

tion

Cha

ract

eris

tics

of D

iter

pene

Res

in A

cid

Met

hyl E

ster

s on

Cap

illar

y G

LC

Col

umns

. Ret

enti

ons R

elat

ive

to M

ethy

l Pim

arat

e.

834 NAVAL STORES

cinylisocupressic acid (found in the foliage of Pinus ponderosa (231) and P. sibirica (232 ). The primary limitation of the nonpolar columns is detector related in that the minimum amount of sample necessary for reliable quantitative data for components a t the 1-2% level can result in overloading the column. Consequently, the data suffers from decreased resolution of some of the closely-eluting major components. One solution is to combine data from analysis on BDS and methyl silicone columns. Cyanosilicone-coated capillary columns are being used in a number of laboratories for resin acid analysis. Such columns have the advantage of being temperature programmable to higher temperatures than BDS columns, but the cyanosilicone columns show the same tendency for overloading as do the methyl silicones. The optimization of efficiency and capacity of the thick-film, wide-bore capillary columns has yet to be achieved (41).

As noted earlier, analysis is more effective with the esters than the free resin acids. Although it has long been known that resin acids will elute from a gas chromatographic column, the only documentation was provided by Phillips for packed columns (303). There is little reason not to use methyl esters for most purposes, but direct analysis of the free acids with the attendant limitations may be of value in process control (302,304 ).

Retention data for resin acid esters are usually expressed by comparing retention with that of the same ester of a specific fatty or resin acid ester. Other approaches have included the use of propyl dehydroabietate with resin acid ethyl esters (50), alkyl phthalates (227), Kovats indices (224) or ECL (equivalent chain lengths with a series of n-fatty acid methyl as reference, 222) .The author has preferred to express retention data with respect to methyl pimarate, the first of the common resin acids to elute from polar and nonpolar columns. The most extensive set of retention data has been reported for over 70 resin acids using a range of six nonpolar to polar liquid phases (223); Table 2 summarizes retention data for methyl silicone and BDS capillary columns from reference 223 along with data for resin acids more recently isolated from pine extractives and distilled tall oil.

GLC has also been applied to the analysis of fumaric and maleic anhydride adducts of resin acids and rosins (packed columns, 277; capillary columns, 278) and for rosin dimers (packed column, 279-281; capillary column, 112). Analysis of rosin dimers is quite complex since strong acid dimerization results in some 40 dimer acids (112). Mono- and dichlorodehydroabietates, found in the effluents from bleaching operations (239), are determined by GLC; retention data for the chlorodehydroabietates have been published for methyl esters on a packed column (282, in the form of a chromatogram) and for the ethyl esters on a capillary column


(50). A chromatogram of a methylated disproportionated rosin as obtained with a methyl silicone-coated capillary column has been published (283).

OTHER TERPENES. The gas chromatographic analysis of turpentine for quality control is covered in Chapter 26. Other pertinent applications of GLC include the analysis of conifer oleoresins and the ring opening products from pinene and carene (284), analysis of terpenes by GC-FTIR (285), monitoring the stereochemical course of ß-pinene hydrogenation (GLC using a-cyclodextrin, 286), analysis of alkylated derivatives of carane and cycloheptane, 287), determination of optical purity of monoterpene olefins by GLC of ketal derivatives (288), and separation of hydroperoxides from the photosensitized oxidation of a monoterpene (limonene, 289). Retention data using the Kovats index has been reported for 20 monoterpene hydrocarbons on SE-30 and Carbowax 20M (290).GLC analysis of turpentines can be accomplished by direct injection of xylem oleoresin (32) and needle oleoresins (42). Retention data for packed columns are available for sesquiterpene hydrocarbons (291,292) and alcohols (293). Information on the gas chromatography of neutral diterpenes in tall oil is given in Chapter 11 and is supplemented by other published retention data for diterpene hydrocarbons on fused silica columns (294). A strategy for quality control in the quantitative analysis of naval stores materials, particularly monoterpenes, has been developed (295).

Quantitation. Composition of individual fatty acids and resin acids in tall oil, tall oil distillation products, and rosins is usually obtained by calculating the normalized peak areas to 100%.It is, of course, necessary that the methodology is valid, such as the procedures used to optimize operating conditions, particularly with attention to injection parameters such as sample size and splitting (capillary columns)(41,225,296). Although the peak-area normalization approach is adequate for many purposes, it is not fully quantitative in that detector responses for individual components can vary by as much as 10%.This situation is recognized for fatty acids in ASTM (297) and AOCS (298) methods and for resin acids ( 4 1 ) ; differences in resin acid response factors have been considered by other investigators, but the data are often of limited value because the resin acids standards were not of sufficient purity.

Obtaining a value for the total amount of fatty and/or resin acids in a sample (with due consideration for any neutral components) would seem to be a simple matter of the sum of the parts. However, this is not readily accomplished in practice because of cumulative problems in the quan-

836 NAVAL STORES

titation of the many minor components. Nevertheless, any attempt to obtain such recovery or throughput data requires an internal standard that is well resolved from any component of the sample.

Choice of an internal standard has been a concern since some of the early research on the GLC of resin acids. For example, Nestler and Zinkel (210) found that the long chain n-alkanes gave anomalous responses with a katharometer detector and were not usable. A hydrocarbon would be expected to be the preferable internal standard because it would be completely inert to the chromatographic process. Hydrocarbons compatible in the retention region of resin acid methyl esters on polyester columns are 24 to 28 carbons in length. With the flame ionization detector (FID) and a packed column (EGSS-X), however, hydrocarbons above C-24 give correction factors with much greater (a factor of 4) relative standard deviation than do fatty acid methyl esters as internal standards (41). Thus, fatty acid esters have been the internal standards of choice; methyl eicosanoate (20:0) for the analysis of oleoresins and gum and wood rosins and methyl heptadecanoate (17:0) for extractives, tall oil, and its distillation products (20:0 is found in these materials). The weight responses for methyl 17:0 and methyl pimarate are essentially equal, but the peak area of methyl pimarate must be multipied by 0.96 when using 20:0; i.e., the weight (detector) response is greater for pimarate. Data must also be corrected for differences in the relative responses of the resin acids (Table 3). Diterpene internal standards, n-propyl dehydroabietate, and methyl Omethyl podocarpate have been suggested as internal standards in the analysis of pulp mill effluents (50).

We and others have attempted to develop recovery or throughput data for a variety of rosins but have found that the throughput of components eluting in the diterpene resin acid ester region have ranged from 80%-

Table 3. Relative Correction Factors for Quantitative GLC of Resin Acid Methyl Esters (41).

Ester (Methyl)

Pimarate Levopimarate Palustrate Isopimarate Abietate Dehydroabietate Neoabietate

Correction Factors for Columns Capillary Packed

BDS SE-30 EGSS-X

1.00 1.00 1.00 1.04 1.03 1.07 1.04 1.06 1.04 1.00 1.00 1.00 1.00 1.00 1.08 1.00 1.00 1.01 1.02 1.02 1.04

24. ZINKEL R e s e a r c h M e t h o d s 837

100%. The lowest values have generally been for gum rosins and the

highest for tall oil rosins. Although we have gained some insight into the

throughput problem by evaluating the potential effects of the presence of

resin acid anhydrides, dimers and neutrals (obtained by both steam dis

tillation or DEAE-Sephadex separation), further research is needed to

obtain a complete material balance and an understanding of rosin

composition.

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