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Anal. Chem. 1902, 5 4 , 174-178
Characterization of Lignin by Gas Capillary Chromatography of Cupric Oxide Oxidation Products John I. Hedges" and John R. Ertel School of Oceanography, Universiv of Washington, Seattle, Washlngton 98 195
Llgnln compounds are hlgh molecular welght phenolic polymers that occur In the woody tissues of vascular plants and In soil and sedlmentary organlc matter. A sensitive, reproduclble method Is descrlbed for the characterlzatlon of llgnlns In untreated plant and geochemical samples contalnlng as llttle as 10 mg of organic matter. The whole sample Is treated wlth alkaline cuprlc oxide at 170 O C to produce slmple Ilgnln-derlved phenols that are extracted wlth ethyl ether and analyzed by gas caplllary chromatography on fused slllca columns. A sutle of up to 11 phenols Is produced that reflects the relatlve concentratlon and plant tlssue sources of llgnlns present In the sample.
used in these previous methods, it alone involves use of a 14C recovery standard and analysis of MeaSi derivatives by gas capillary chromatography with fused silica columns. This method, although entirely suitable for plant tissue samples, is designed specifically for the characterization of small amounts of lignin in complex organic mixtures such as those found in soil and sediment samples (17, 18). As little as 10 mg of total organic matter in untreated solid samples or aqueous solution is needed. Both carbonyl and carboxyl substituted phenolic oxidation products are precisely quantified to yield detailed compositional information reflecting plant tissue sources and concentrations (6, 19).
EXPERIMENTAL SECTION Lignin compounds are phenolic polymers that occur as major constituents of the cell walls of vascular plants (I). As a result of their natural abundance, wide distribution, and resistance to microbial degradation, lignins are also commonly found in soil and sedimentary organic matter (2-6). Lignin polymers in plant tissues, soils, and sediments are not amenable to direct chemical analysis without prior isolation. However, lignin in such samples can be characterized by chemical degradation to release small molecules that can extracted from the reaction mixture and quantified by a variety of chromatographic techniques (1, 7). Mild oxidation with nitrobenzene or cupric oxide is one of the most commonly used methods of degradative chemical analysis. These oxidizing agents yield 25-75% of the treated lignin in the form of simple phenols that preserve many of the chemical characteristics of the structural units in the original polymers (8). Since its introduction by wood chemists (9) nitrobenzene has been the reagent of choice for studies of lignin structure. However, nitrobenzene produces a number of organic byproducts which can interfere with phenol analysis (8).
For this reason cupric oxide is often chosen as a more suitable oxidizing agent, especially for characterizing small quantities of lignin. CuO produces lignin-derived phenols in overall yields comparable to those obtained with nitrobenzene (8). The compositions of the oxidation product mixtures are also similar, with the exception that CuO produces ketonic phenols (Table I) in addition to the aldehydic and acidic analogues yielded by nitrobenzene. Aldehydic phenols are the predominant products of both oxidizing agents and often are used exclusively for characterizing lignins in plant and geochemical samples (1,2, 10, 11). A wide variety of derivatizing agents can be used to convert lignin oxidation products to volatile, more chemically stable forms for gas chromatographic analysis. Of these reagents trimethylsilyl (Me,Si) donors are particularly useful because they allow rapid, one-step derivatization of both phenolic and carboxyl functional groups with a wide variety of commercially available reagents (12, 13). A number of procedures have been published for characterizing lignin-bearing materials by gas chromatographic analysis of CuO oxidation products (e.g., ref 5 , 14-16). Although the procedure described here incorporates techniques 0003-2700/82/0354-0174$01.25/0
Cupric Oxide Oxidation. Soil, sediment, and plant samples are freeze-dried and ground to pass a 42-mesh (0.351 mm) sieve. If desired, lipids can be extracted prior to analysis. The CuO oxidation is carried out in 10-mL Monel "minibombs" which are sealed with Teflon-lined screw caps (Figure 1). Four such minibombs can be loaded into a commercially available 2 O O - d Parr bomb (Model 4753) allowing oxidation of up to four samples at once. The minibombs are opened and closed with a wrench and block fitted with pins that match holes drilled in the screw cap and base. After introduction of a radioactive tracer (see later discussion) each minibomb is charged with 1.00 g of CH2C12-extractedCuO (powdered), 25-100 mg Fe(NH4)2(S04)2.6H20 (used as an O2 scavenger), 7.0 mL of 8% (wt/wt) aqueous NaOH, and a small stainless steel ball agitator. Typical sample loadings are 25 mg of plant material, 50 mg of humic acid, and 0.1-2.0 g of soil or sediment (depending on organic matter content). Individual minibomb charges are weighed into open glass vials that are loaded along with the open minibombs and 200-mL bomb pieces into a N2-purgedglovebox one day before analysis. The following day the solid components are added to the individual minibombs and the NaOH solution (preboiled and stored under N2)is introduced via an autopipettor that is also stored in the glovebox. In this manner the bombs are sealed without introduction of O2 After the four sealed minibombs are loaded into the 200-mL.bomb, 1.00 g of CuO, 25-100 mg of Fe(NH4)2(S04)2*6H20, and sufficient 8% NaOH solution to just cover the top minibomb are added before sealing to produce similar pressure and chemical conditions inside and outside of the minibombs. The CuO oxidation is carried out at 170 "C on a platform shaker. The 200-mL. outer bomb is heated in a snug brass cylinder that is wrapped with electrical heating tape and asbestm insdating cloth inside on aluminum tape covering. The reaction temperature is maintained with a proportional temperature controller (Cole Parmer Model C-2155-20)fitted with an armored platinum temperature probe that is mounted in an insulated pocket in contact with the brass heating sleeve. The bomb can be heated from room temperature to 170 OC in approximately 20 min. The oxidation is terminated after 3 h with a timed switch and the large bomb is immediately cooled under running tap water. The contents of each minibomb are washed with 1 N NaOH into a 50-mL centrifuge tube and rotated at about 1200 ppm (-250 g) for 10 min in an International Clinical centrifuge (Model CL45387M-14). After the supernatant is decanted and saved, the sediment is washed twice with 20 mL of 1 N NaOH (after dispersion with ultrasonic vibration) and centrifuged as before. The combined basic extracts are acidified to pH 1with 6 N HCl and extracted successively with three 20-mL volumes of distilled ethyl ether that has been previously treated with a saturated aqueous 0 1982 American Chemlcal Society
ANALYTICAL CHEMISTRY, VOL. 54, NO. 2, FEBRUARY 1982
175
Table I. Common CuO Oxidation Products of Lignin
\ R5
A = CHO
D
B = COCH,
E=H
C = CO,H
E’ = OCH,
structure
= ~~~T~s-CH=CHCO,H
compound
R,
R3
R,
mol wt plain Me,&‘
p-hydroxybenzaldehyde
A B C A B
E E E E E E F F F E E
E E E E‘ F F F F F E F
122 136 138 152 166 168 182 196 198 164 194
p-hydroxy acetophenone
p-hydroxybenzoic acid vanillin. acetovanillone vanillic acid syringaldehyde acetosyringone syringic acid p-coumaric acid ferulic acid
C
A B C D D
194 208 282 224 238 312 254 268 342 308 338
plant sources b g A
G
a
*
*
* * * * * *
* *
* * * * *
* *
* * * *
* * * *
* *
* *
* *
G = gymnosperm woods, g = nonwoody gymnoa Molecular ,weight of the trimethylsilyl derivative. b Abbreviations: sperm tissues, A = angiosperm woods, a = nonwoody angiosperm tissues. The asterisk indicates typical production at greater than 1 wt % of the total phenolic yield (17, 19). ! 40 mm -*-
[a-
I
I
O
(ai
Y
\
i
12
I
i
’P
2mm thick t13~m-
Figure 1. Scale drawing of a “minibomb”. The 2-mm thick gasket (stippled) in the bomb cap is a solid Teflon dlsk. L
solution of Fei(NH4)2(804)2.6H20 to remove peroxides. The combined ether extracts are passed through an anhydrous Na2S04 column and the volume of ether is reduced to 1-2 mL by vacuum rotoevaporation. Most water present in the sample at this point can be removed with a small amount of anhydrous Na2S04 The product is then transferred with an additional 1-2 mL of predried, Fe2+-treatedether to a preweighed 1-dram vial with a Teflon-lined screw cap. The ether is then removed under a stream of N2after which the total reaction product is weighed and stored in a refrigerator until analysis. Only reagent grade chemicals are used in this procedure. All glassware is washed in Na3P04-treatedwater and rinsed successively with distilled water, alkaline methanol, distilled water and methanol. Gas Chromatography. The solid oxidation product is dissolved in pyridine (freshly distilled) containing 0.333 rg/pL of ethylvanillin a8 an internal GC standard. From 10 to 200 & of internal standard solution is typically used, depending on the expected yield of lignin-derived phenols. From 50 to 200 pL of Regisil plus 1% trimethylchlorosilane (Regis Chemical Co.) is then added to obtain approximately equivalent volumes of pyridine and silylating agent. The resulting solution is heated to 68 OC for 10 min to complete formation of the trimethylsilyl (Me3Si) derivatives of the GC internal standard and acidic reaction products. The solution is cooled to room temperature and immediately analyzed by gas chromatography. A Hewlett-Packard 5700A gas chromatograph fitted with a flame ionizationi detector is used for all analyses. The GC injector is modified for ‘^split”injections onto a capillary column and has a glass liner that is partially filled with silylated glass beads. The glass beads andl the fore end of the capillary column sometimes require daily renewal in order to maintain optimal chromatographic performance when analyzing samples with a high proportion d nonvalatile oxidation products. Chromatographic analyses are routinely made with a 30 m by 0.25 mm i.d. fueled silica capillary column (J & W Scientific, Inc.)
Y
JL
TEMPERATURE IPCI
Figure 2. Gas chromatographic traces of (a) a standard mixture of Me,Siderlvatlzed phenols and carboxyllc acids and (b) the Me,Si derivatlves of the CuO oxidation products from a recent freshwater sediment (Lake Washington sample 0.06). Gas chromatographic equipment and conditions are described in the text. Average phenol yields for sediment sample 0.06 are given in Table 11. Identifications (for unsilylated precursors): 1, benzoic acid; 2, p-hydroxybenzaidehyde; 3, p-hydroxyacetophenone; 4, vanillin; 5, rn-hydroxybenzoic acid; 6, ethyivaniilin (GC internal standard); 7, acetovanillone; 8, p-hydroxybenzoic acid; 9, syringaldehyde; 10, acetosyrlngone; 11, vanillic acid; 12, 3,5dihydroxybenzolcacid; 13, syringic acid; 14, trans-p-coumaric acid; 15, trans-ferulic acM; 16, n-heptadecanolc acid. X and Y in trace (a) lndlcate the respective elution points of cis-p-coumaric acid and cis-ferulic acid.
coated with SE-30 (methylsilicone)liquid phase. This liquid phase provides complete resolution of the MeaSi derivatives of all the major lignin-derived phenols (Figure 2a) and minimum interference from other common oxidation products. The injection is split at a ratio of approximately 1/50 with an initial column
176
ANALYTICAL CHEMISTRY, VOL. 54, NO. 2, FEBRUARY 1982
flow rate of about 1.5 cmg/min of helium. Column temperature is programmed from 100 "C to 270 "C at 4 "C/min with no initial delay. The FID analog signal is recorded and processed by a Hewlett-Packard Model 3390A integrator. Both injector and detector are maintained at 300 "C. Response factors of all the other compounds indicated in Figure 2a are routinely determined relative to the GC internal standard (ethylvanillin) by injections of this standard mixture at the beginning of GC analysis and approximately every fourth sample thereafter. Silylated standard mixtures are prepared volumetrically from a 1 (mg/mL)/component master standard (in pyridine). Silylated standard solutions are typically stable, but should be monitored for loss of cinnamyl phenols (see later discussion). The synthetic chemical, ethylvanillin, is used as a GC internal standard because it elutes with minimal interference from other sample components and is chemically similar to the quantitatively predominant aldehydic lignin oxidation products. 14C Recovery Standard. Uniformyl ring labeled [14C]-phydroxyacetophenone (Table I) is used to estimate the overall recovery of all lignin-derived oxidation products. The use of this procedure is based on the previous observations (Hedges, 1975) that (a) measured recoveries of radioactive tracer and unlabeled p-hydroxyacetophenone following their CuO oxidation agree within analytical error (91% and 94%, respectively, for an experiment in which 10 mg of compound was so treated) and (b) that the extraction efficiency of p-hydroxyacetophenone is representative of the other major lignin oxidation products in Table I. Approximately 2.5 X pCi (1.3 pg) of radioactive tracer is introduced in 100 pL of toluene to each minibomb before charging and the solvent is completely removed under Nz. After derivatization of the oxidation product for injection into the GC, triplicate 1 4 % aliquots are transferred with a 1O-pL syringe to 20-mL liquid scintillation vials and counted in a "cocktail" containing 5.0 g of PPO and 0.1 g of POPOP (Packard Instrument Co.) per liter of scintillation grade toluene. These preparations are analyzed in a Packard Model 3310 liquid scintillation spectrometer for a total of 40000 counts vs. equivalent volumes of the parent spiking solution and similarly prepared blank solutions, to determine background-corrected average percentages of recovery. Use of the 14Crecovery standard necessitates careful venting of the GC splitter and detector effluents during analysis. R E S U L T S AND DISCUSSION Common Lignin-Derived Phenols. The chemical structures, molecular weights, and plant tissue sources of 11 major lignin-derived CuO oxidation products are given in Table I. These phenols can be divided into four different structural families. The p-hydroxyl, vanillyl, and syringyl families consist of three phenols each, an aldehyde, ketone, and carboxylic acid, which differ only in substitution on the carbonyl carbon. The cinnamyl family consists of two phenols with trans propenoic acid substitution. The Me3& derivatives of all 11phenols give prominent molecular ions when analyzed by electron ionization (70 eV) mass spectrometry. Woody and nonwoody tissues of gymnosperms (nonflowering vascular plants) and angiosperms (flowering vascular plants) each yield a distinctive combination of phenols from the four structural families (Table I). With rare exceptions for the cinnamyl phenols (19),either all or none of the members of an individual phenolic family are produced by CuO oxidation. Thus, distinctive multicomponent chemical signatures are obtained that are particularly useful for the characterization of the lignin components of complex organic mixtures typically found in most geochemical samples. Of these 11phenols only the p-hydroxyl compounds are known to have quantitatively important nonlignin sources that sometimes limit their use as geochemical tracers (17). A gas chromatogram of a standard mixture containing the previously discussed lignin-derived phenols and a number of common nonlignin oxidation products is shown in Figure 2a. A chromatogram of the CuO oxidation product mixture from a freshwater sediment (Figure 2b) illustrates the compositional
complexity of a typical geochemical sample. The predominance of aldehydes over the corresponding ketones and carboxylic acids in the p-hydroxyl, vanillyl, and syringyl phenol families (Figure 2b) is typical of the CuO oxidation products of lignins in plant tissues and most geochemical samples (17, 19). Analytical Sensitivity a n d Reproducibility. The sensitivity of the flame ionization detector for individual Me3Si-phenol derivatives is approximately 0.1 ng. Assuming a minimum sample volume of 50 pL, a maximum injection volume of 2 pL, and a ' / m split of the injected volume, this sensitivity corresponds to a minimum detectability of about 0.1 pg of an individual phenol in an oxidation product mixture. Procedural blanks (containing all reagents, but no sample) routinely yield less than 1Mg/sample for each of the phenols in Table I except of p-hydroxyacetophenone which is introduced as a recovery standard. If necessary, the weight of the radioactive tracer (calculated from its specific activity) can be subtracted from the total yield of p-hydroxyacetophenone after recovery correction. The other major components of a typical blank are normal fatty acids with 12-18 carbon atoms per molecule that usually are recovered in amounts less than 2 pg each per sample. The radioactivity of aliquots of the silylated oxidation product mixtures can be determined with an average precision (% sample standard deviation) of *l%(based upon triplicate analyses of 1-3% aliquots from 25 samples of different compositions and initial volumes). One-microliter volumes of the radioactive spiking solution can be prepared and counted with similar precision. Procedural losses of the radioactive tracer due to volatilization and effects of the pyridine-Regisil solvent system on the counting efficiency of sample aliquots do not occur to a measurable extent. The reproducibility of the analytical technique for the 11 lignin-derived phenols in Table I was tested by duplicate analyses of five freshwater sediment samples which differed widely in organic composition (Table 11). Percent mean deviations do not exceed *15% for the yield of an individual phenol from any sample. Average deviations for individual phenols are less than 10% and average 5.% overall. Absolute yields (yg/g dry sediment) for individual phenols from different samples in this set vary by at least 1order of magnitude. There is, however, no apparent relationship between phenol yields and analytical reproducibility. In actual application absolute yields of lignin-derived phenols are used to generate a variety of extensive (yield) and intensive (ratio) parameters that allow concise descriptions of lignin content and composition (6, 19). Table I11 gives percent mean deviations for 15 lignin parameters calculated from the previously discussed set of five duplicate analyses (Table 11). Only the acid/aldehyde ratio for the p-hydroxyl phenol family has an average mean deviation greater than 10%. This large variability is due primarily to poor analytical precision for p-hydroxybenzaldehyde (Table 11) that results from incomplete separation of a large preceding chromatographic peak (e.g., Figure 2b). Weight percentages of organic carbon (used to calculate P, V, S, C, and their sums) were determined for this group of sediments with an average reproducibility of k2% (sample standard deviation) that is included in the calculations in Table 111. Special Considerations. A number of precautions must be taken during sample preparation and analysis to avoid experimental artifacts that can occur. One necessary precaution is that all ethyl ether that contacts the sample must be freshly treated with Fez+ (see Experimental Section) to remove peroxides. Syringaldehyde in particular is sensitive to peroxides, with percentage losses increasing with decreasing sample size (Figure 3). Although this problem can be avoided
ANALYTICAL CHEMISTRY, VOL. 54, NO. 2, FEBRUARY 1982
177
~
Table 11. Average Phenol Yields (pg/g Dry Weight) from Duplicate Analyses of Rve F’reshwater Sediments‘ compound
0.06
3.9
p-hydroxybenzaldehyde p-hydroxyacetophenone p-hydroxybenzoic acid vanillin acetovanillone vanillic acid syringaldehyde acet osyringone syringic acid p-coumaric acid ferulic acid
55.8 f. 7.6 16.9 :t 1.7 76.7 i 3.8 214f 1 87.2 f: 0 123 f 1 108 f 5 44.4 .F 0.4 35.8 t 0.8 36.3 f 0.1 55.2 f 2.7
128 f 1 2 33.3 f 1.2 133 fr 0 279 f 27 104 f 7 127 i 5 251 i 31 108 f 10 76.4 f 1.9 70.0 1.6 75.2 i: 6.0
mean
sample 7.4
%
109 f 3 29.4 f 0.5 118 +- 0 240 f 2 92.9 f 1.4 114 f 0 204 2 2 88.0 f 3.0 65.4 % 1.0 78.3 % 0.8 79.2 f 7.1
8.9
11.0
deviation
61.5 % 7.9 9.10 f 0.5 26.1 f 0.4 50.9 f 1.0 19.0 f 1.4 20.2 % 2.2 50.6 f 6.6 22.9 f 1.1 12.5 f 0 82.7 f 2.0 20.3 f 1.0
3.09 i 0.33 1.13 f 0.08 3.47 0.21 11.6 i 0.5 3.51 f 0.29 4.55 f 0.33 4.70 f 0.40 1.72 f 0.02 1.48 f 0.04 1.67 f 0.10 0.84 f 0.12
9.9 5.6 2.6 3.4 4.9 4.6 7.9 4.0 1.9 2.4 6.1
a Intervals about the mean are sample mean deviations: (EIF - Xi[ In). The average % recovery for the five pairs of duplicate analyses was 7 1 i 4 (+1 standard deviation, n = 3 0 ) as indicated by the I4Crecovery standard.
Table 111. Reproducibi1,ity of Individual Lignin Parameters‘ parameter Pb
V S C
3.9
!2.3 L.5 13.4 4.8
5.3 8.3 10.4 3.5 9.3 8.6 8.7 7.7 2.6 2.4 5.3 10.2 9.5 6.5 9.7
‘2.0
hC Ad
‘2.4 0.4 0.8 ‘2.9 ‘2.2
c 6e x8f
P/V
s/ v
c/vg
0.06
F/C (AdlAl)ph (Ad/Al)v (Ad/Al)s
2.3 ~4.6
ld.4 0.8 ‘7.5
samvle 7.4 1.6 0.4 1.8 4.7 0.7 0.3 0.1 0.8 1.8 1.9 4.3 7.9 3.2 1.1 0.0
8.9
11.0
av
8.6 5.5 9.2 3.1 7.3 5.7 7.3 5.4 14.3 3.7 2.2 4.0 5.0 8.9 14.2
6.2 2.7 2.9 12.1 1.2 2.2 1.8 1.6 3.8 5.0 7.7 8.0 17.0 12.8 4.8
4.8 3.7 5.5 5.6 4.1 3.8 3.6
I
/
I
3.3 5.1 3.0
4.4 6.9 10.6 6.0 7.2
Percent sample mean deviation. b P , V, 8,and C = total mg of p-hydroxyl, vanillyl, syringyl, and cinnamyl phenols, respectively, produced from 100 mg of organic carbon. h = V + S. A = V + S + C. e Total milligrams of vanillyl and syringyl phenols produced from 10 g of dry sample. f As before including cinnamyl phenols. g Weight ratio ferulic acid to p-coumaric acid. h Acid/ aldehyde weight ratios for p-hydroxyl, vanillyl, and syringyl phenols, respectively. a
0‘
,b
I 100
!
Flgure 3. Effect of Fe*+ treatment on the relative yield of syringaldehyde in the phenolic mixture produced by CuO oxidation of wood from the palm tree, Phoenix dactybferra(20).
Table IV. Phenol Compositional Changes Following SilyIationa
--
compound by use of some other extraction solvenlt such as dichloromethane (2), redluced recoveries of acidic oxidation products can occur with halocarbon solvents (20). A second possible problem is slow loss of silylated carbonyl compounds from the pyridine-Regisil solutions of some oxidation product mixtures after preparation for gas chromatography. These losses occur to comparable extents with the p-hydroxyl, vanillyl, and syringyl phenol families and are always more pronounced for aldehydes than the corresponding ketones (Table IV). Corresponding losses from standard solutions of silylated phenols have not been observed even after months of storage. The rate of phenol loss from sample solutions is highly variable but appears to be most rapid for oxidation product mixtures which contain small relative amounts of lignin-derived phenols. Carbonyl phenol losses are always attended by appearances of new GC peaks, suggesting that a t least a portion of the original compound is converted into volatile secondary products. Similar behavior has been observed previously using a different silylating agent (Silyl-8, Pierce Chemical Co.) under similar conditions (20). The loss of carbonyl compounds was
I
1000
Sample Weight h g )
p-hydroxybenzaldehyde p-hydroxyacetophenone p-hydroxybenzoic acid vanillin ethylvanillin acetovanillone syringaldehyde acet osyringone syringic acid
initi9 final ratio ratiob to to vanillic vanillic % acid acid changeC 0.46 0.24 1.21 1.00 0.42 0.61 0.67 0.46 0.52
0.33 0.23 1.24 0.69 0.27 0.55 0.42 0.43 0.56
-34 -12 t2 -32 -36 -9 -30 -6 t6
a A total of 40 days elapsed between initial silylation and analysis and the second analysis. Height ratios measured by hand to the nearest millimeter. C Calculated relative to the initial ratio. This sample is “superoxidized” as indicated by relatively high initial acid/ aldehyde ratios within all three phenol families.
found to result from their reaction with formamide to form a volatile Schiff base addition product. We suspect a similar mechanism is responsible for carbonyl losses using the present procedure for silylation but as yet have not identified the secondary products. Immediate analysis of oxidation product
178
Anal. Chem. 1982, 5 4 , 178-181
mixtures following silylation and use of a GC internal standard, ethylvanillin, that chemically resembles the predominant aldehydic oxidation products of lignin, both minimize artifacts due to carbonyl loss and allow excellent analytical precision for these compounds (Table 11). If necessary for particularly difficult samples, an additional noncarbonyl GC internal standard can be used so that the extent of aldehyde loss can be estimated from changes in its ratio to ethylvanillin. A third potential problem that is also quite variable in its impact is geometric isomerization of the cinnamyl phenols in silylated standard and sample solutions. The p-coumaric and ferulic acid produced by lignin oxidation and most commercially available counterparts are essentially pure trans geometric isomers. However, for some as yet unknown reason, slow isomerization to the corresponding cis form occurs for both compounds in some sample and standard solutions following silylation. Ferulic acid typically isomerizes much faster than p-coumaric acid. Geometric isomerization is too slow to have a measurable affect when GC analysis of sample oxidation product mixtures is performed immediately after silylation. However, isomerization effects can be significant (up to 10% /day) for some standard mixtures whereas others may be stable for weeks. Routine monitoring of silylated standard solutions for conversion of trans cinnamyl phenols to earlier eluting cis isomers (Figure 2a) is, therefore, prudent. The final problem that is sometimes encountered in the described analytical procedure is “superoxidation” of the sample as indicated by consistently high acid/aldehyde ratios within the p-hydroxyl, vanillyl, and syringyl phenol families (e.g., Table IV). These high ratios primarily reflect conversion of aldehydes to the corresponding carboxylic acids. Although such conversions in themselves have little effect on the lignin parameters P, V, and S or their derivative parameters (Table 11),other compositional artifacts such as preferential loss of syringyl phenols and overall decreased phenol yields also occur which are not corrected by summation within phenol families. Superoxidation can result from introduction of O2into the minibomb during charging or from elevated reaction tem-
peratures (20). The problem is most likely to occur in samples containing little organic matter.
ACKNOWLEDGMENT The authors thank P. Parker, T. C. Hoering, and R. Carpenter for numerous contributions.
LITERATURE CITED Sarkanen, K. V.; Ludwig, C. H. “Lignins”; Wiley: New York, 1971. Morrison, R. I. J. Soil Sci. 1963, 74, 201-216. Leo, R. F.; Barghoorn, E. S. Science 1970, 768, 582-584. Schnitzer, M.; Khan, S. U. “Humic Substances in the Environment”; Marcel Dekker: New York, 1972. (5) Dormaar, J. F. Can. J. SollSci. 1979, 59, 27-35. (6) Hedges, J. I.; Mann, D. C. Geochlm. Cosmochim. Acta 1979, 43, 1809- 18 18. (7) Pearl, 1. A. “The Chemistry of Lignin”; Marcel Dekker: New York,
(1) (2) (3) (4)
1967.
(8) Chang, H.-M.; Aiian, G. 0.I n ”Lignins”; Sarkanen, K. V., Ludwig, C. H., Eds.; Wiley: New York, 1971; pp 433-485. (9) Freudenberg, K.; Lautsch, W.; Engler, K. Chem. Ber. 1940, 73, 167. (10) Farmer, V. C.; Morrison, R. I . Geochlm. Cosmochlm. Acta 1964, 28,
1537-1546. (11) Gardner, W. S.; Menzel, D. W. Gsochim. Cosmochim. Acta 1974, 38, 8 13-822. (12) Nelson, P. F.; Smith, J. 0.Tappl 1966, 4 9 , 215-217. (13) Knapp, D. R. “Handbook of Analytical DerivatizationReactions”; Wiiey: New York, 1979. (14) Hartiey, R. D. J. Chromatogr. 1971, 5 4 , 335-344. (15) Schnitzer, M. SoilBlol. Biochem. 1974, 6 , 1-6. (16) Hayatsu, R.; Winans, R. E.; Scott, R. G.; McBeth, R. L.; Moore, L. P.; Studier, M. H. Sclence 1980, 207, 1202-1204. (17) Hedges, J. I.; Parker, P. L. Geochlm. Cosmochlm. Acta 1976, 4 0 ,
1019-1029.
(18) Ugolini, F. C.; Reanier, R. E.; Rau, G. H.; Hedges, J. I. Soil Sci. 1981,
737,359-374. (19) Hedges, J. I.; Mann, D. C. Geochlm. Cosmochlm. Acta 1979, 4 3 , 1803-1 807. (20) Hedges, J. 1. Ph.D. Dissertation, University of Texas at Austin, Austin, TX, 1975.
RECEIVED for review August 18, 1981. Accepted October 20, 1981. Acknowledgment is made to the donors of the Petroleum Research Fund, administered by the American Chemical Society, for partial support of this research. This research was also funded in part by NSF Grants OCE-7818260 and OCE-8023970. This is Contribution No. 1238 from the School of Oceanography, University of Washington, Seattle, WA.
Separation of Cationic Metal Chelates of 1 , lO-Phenanthroline by Liquid Chromatography Jerome W. O’Laughlln Department of Chemistry, Universi@ of Missouri, Columbia, Missouri 652 1 1
Ion palr, hlgh-performance llquld chromatographic separatlon of the Inert 1,lO-phenanthrollne chelates Fe(phen):+, Ru(phen)32+, and Nl(phen),’+ on polystyrene-dlvlnylbenzene polymer based columns Is reported by use of acetonltrllewater-perchlorlc acld mlxtures as the moblle phase. The extenslon of thls technique to the separation of the lablle chelates Zn( phen),”, Co( phen);’, Cd( phen),”, and Cu(phen),”, when the moblle phase Is l o 4 M In the ligand and LICIO, Is used In place of HCIO, as a source of the palrlng Ion, Is shown to be feaslble. The effects of moblle phase parameters on retention and resolutlon are reported on several dlfferent types of columns.
The separation of the 1,lO-phenanthroline chelates Ni0003-2700/82/0354-0178$01.25/0
(phen):+ and Ru(phen):+ from Fe(phen):+ but not from each other by ion-pair, high-performance liquid chromatography has been previously reported (1). The separation of all three of the above chelates from each other and the extension of this technique to the separation of the labile 1 , l O phenanthroline chelates of Zn(II), Cd(II), Co(II), and Cu(I1) on fi-Partisil-SCX(cation exchange) and the Hamilton-PRP-1 (polystyrene-divinylbenzene bead) columns with the perchlorate ion as the pairing ion is reported in the present paper. The effects of mobile phase parameters, volume percent acetonitrile, pH, the perchlorate ion concentration, and the 1,lO-phenanthroline concentration on the retention volumes are reported. The successful separation of the labile chelates when the mobile phase was kept 10”. M in l,l0-phenanthroline is shown. The retention volumes were found to decrease exponentially with an increase in the perchlorate ion con@ 1982 American Chemical Society
