Characterization of Lignosulfonates by Ultraviolet Spectrometry. Direct

taking place n-ithin the growth period examined. The present obsei*vations challenge the conventional idea that alkaloids are mere end-products of metabolism. Birecka et al. (3) obtained similar results for older plants a t flowering and seedpod formation, where high physiological activity might be esoected. Both sets of observations indicate that the most rapid alkaloid metabolism takes place when the plant is physiologically most active, and suggest that alkaloids may play a more active role in the physiology of the plant than Iias hitherto been suspected.

ACKNOWLEDGMENT

( 5 ) L)awson, K. F.,Lkhm.b ’ m ~ t t i o l . 8,

The authors are grateful to Hugh A. White for assistance in the analyses.

(6) Lee, K.-T., Nature 188,65 (1960). (7) Mattocks, A. R., Ibid., 191, 1251

LITERATURE CITED

( 8 ) Mothes, K., Ann. Rev. Plant Physiol. 6 , 393 (1955).

(1) Birecka, H., Kalborczyk, E., Bull. Acad. Polon. Sci. Sdr. Sci. Biol. 9, 401 (1961). (2) Birecka, H., Rybicka, H., SciborMarchocka, A., Bcta Biochim. Polon. 6 , 2 5 (1959). (3) Birecka, H., Szgmahka, A., SciborMarchocka, A., Acta SOC.Boian. Polon. 29,369 (1960). (4) Cromwell, B. T., “Modern Methods of Plant Analysis,” Paech, Tracey, eds., p. 367, Springer-Verlag, Berlin, 1955.

203 (1948). (1961).

(9) Reifer, I., Niziokek, S., Bull. Acad. Polon. Sci. Sdr. Sci. Biol. 7, 485 (1959). (10) Tompsett, S. L., ! c i a Pharmacol. Toxicol. 18, 414 (1961,.

RECEIVEDfor review August 2, 1963. Accepted October 8, 1963. Part of this paper was presented to the meeting of the Canadian Society of Plant Physiologists, Winnipeg, June 1963. A grant-inaid of this research from the National Research Council of Canada is gratefully acknowledged.

Downloaded by UNIV OF CAMBRIDGE on August 23, 2015 | http://pubs.acs.org Publication Date: January 1, 1964 | doi: 10.1021/ac60207a066

Characterization of Lignosulfonates by Ultraviolet Spectrometry Direct and Difference Spectrograms ARTHUR S. WEXLER Dewey and Almy Division, W. R. Grace

b The ultraviolet dkect and difference spectrograms of alkaline and neutral (or acid) solutions of lignosulfonates are valuable in interpreting the chemistry of these Substances. The use of an ultraviolet double-beam recording spectrophotometer to obtain recorded spectral charts of both the direct and differential spectrograms greatly facilitates analysis and study of these and related substances. The direct spectrograms are useful in establishing the gross features of the material. The differential s(:lectrogram is of value in determination of the aromatic hydroxyl content and is also a characteristic physicochemical property of the material. spec$,rometry has been extensively employed in the study of the chemistry and molecular structure of the lignins and 1 gnosulfonates (12, 16-18). The absorption maximum near 280 mp of aqueous or alcoholic solutions of these substances has been frequently cited as supporting evidence of the presence of guaiac.jl, syringyl, and related structures mi th a predominance of aromatic methoxyl over aromatic hydroxyl groups (1, 4, 16, 17). More recently, the differential absorption obtained by manually plotting the difference between the absorptivities of the ionized (in alkaline solution) and nonionized (neutral or acid solution) forms of lignins and ,ignosulfonates has been utilized in studies of molecular structure (2, 3, 8) anil in determination of aromatic hydroxyl content ( 7 ) . LTRAVIOLET

Co., Cambridge, Mass. Discrepancies betrreen the spectroscopically determined aromatic hydroxyl content and the values found by chemical methods hare been reported (6J

Practically all the reported investigations employing ultraviolet spectrometry have been based on single-beam, manually operated, nonrecording instruments. Much progress could be made in spectrophotometric studies of these and related substance by use of a high quality recording ultraviolet doublebeam spectrophotometer to obtain direct and differential spectrograms for analysis and interpretation. The interpretation of recorded direct and differential ultraviolet spectrograms of softwood lignosulfonates and the technique of obtaining such recorded data are the major topics of this paper. Both the direct and differential spectrograms can be recorded on the same spectral chart for ready comparison, analysis, and interpretation. The aromatic hydroxyl content can be determined using a base line technique in the differential spectrograms. Certain definitions are helpful in presentation of data. The differential spectrograms of lignosulfonates display a strong peak at about 250 mp and a weak peak at about 300 mp. Minima are found near 229 and 278 mp. The following symbols are used in this paper : A 250 followed by absorptivity or by mp. Strong peak maximum a t about 250 mp in the differential spectrogram. 4 300. Keaker peak maximum a t about 300 mp in the differential spec-

trogram. This peak is 0.4 to 0.6 as strong as the A 250 peak. A mp. Spacing in millimicrons between the two peak maxima in differential spectrogram. This value is about 50 mp in softwood lignosulfonates. EXPERIMENTAL

Procedure. A Beckman DK-2 ultraviolet recording double-beam spectrophotometer was employed in the linear absorbance mode as follows: Scan speed Gain Slit at 280 mp Time constant

160 mp, in 6 minutes

140

0.06 mm. 0.2

Peak maxima and minima were spot-checked with a Beckman DU single-beam spectrophotometer. Results agreed within 1 to 2% of the measured absorbance. Wavelengths were checked and calibrated with a mercury lamp. Reagents. Solutions of lignosulfonates were prepared as follows for ultraviolet measurements: Exactly 0.2000 gram of sample was transferred to a 100-ml. volumetric flask, dissolved in distilled water, and adjusted to volume a t room temperature (usually about 23’ C.). Second dilutions were made by pipetting exactly 5 ml. with a delivery pipet into each of two volumetric flasks. To one flask was added 10 ml. of 1.ON potassium hydroxide; to the other, 10 ml. of 1.ON hydrochloric acid solution. Adjustments to final volumes of 100 ml. were made with distilled water. In this way identical concentrations of 100 p.p.m. in 0.1N alkali and in 0.1N acid were prepared for ultraviolet measurement. Matched 1-cm. silica cells were VOL. 36, NO. 1 , JANUARY 1964

213

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essed sulfite waste liquor dried material obtained from Marathon, Rayonier, Crown-Zellerbach, Lignosol, and other commercial sources. Most of the samples were calcium, sodium, or ammonium salts. Absorptivities are expressed in liters per gram per centimeter-i.e., litersgrams-i-centimeters-l.

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QUANTITATIVE INTERPRETATION OF SPECTROGRAMS

A typical ultraviolet spectrogram of a softwood-type lignosulfonate is shown in Figure 1. Curves B and A are tracings of 331/p p.p.m. concentration of a crude lignosulfonate preparation in 0.1N acid and alkali, respectively. The shift of the peak maximum a t 280 mp in curve B to 282 mp in curve A , accompanied by a moderate increase in intensity, is characteristic of softwoodtype lignosulfonates. Curve Cis a typical difference spectrogram or A curve obtained by scanning with the alkaline 33’/1 p.p.m. solution in the sample beam and the acidified solution of identical concentration in the reference beam. The presence of peaks a t 250 and 300 mp is typical of lignosulfonates. Some variability is observed in the scans a t wavelengths below A240 mp due to large variations in the slit widths as the instrument scans in a region of low energy input and low detector sensitivity. The reproducibility of successive dilutions on the same and on two successive days is illustrated in Table I. In each example the peak height was measured in millimeters from a zero tracing of the same cells filled with appropriate solvent, except for the results marked “base line,’’ where the peak was measured from a base line connecting the minima a t A229 and 11278 mp. A typical base line is illustrated by the dashed line in Figure 2. Figure 2 is a reproduction of a differential spectrogram a t the 100-p.p.m. level of the same lignosulfonate shown in Figure 1, The base line measurement

WAVELENGTH, m j

Figure 1.

Direct and difference spectra :p.p.m. in 0.1 N KOH

;-y c-..-

:p.p.m. In 0.1 N HCI Difference spectrum with A in sample beam and B in reference beam

employed for most measurements. In all cases the same pipet was employed for the final dilutions in preparation of samples and the same drainage times were taken. For scanning purposes, the alkaline solution was placed in the sample beam and the acidified solution in the reference beam. R e producibility of dilutions was usually within 2% as estimated by absorbance measurements. Repeat scans in the same cells usually reproduced at all points well within 0.01 absorbance unit. There was no evidence of deterioration of either stock solutions or final dilutions within %-hour holding periods in either acid or alkaline solutions, or of specimens due to exposure t o ultraviolet light during a scan. Lignosulfonate preparations were usually obtained as dry powders and were diluted without further treatment. No

Table 1.

Wavelengths 10/15/62 10/15/62 10/16/62 10/16/62 Ranee MG. on chart

5%

of a softwood lignosulfonate

correction was made for moisture content of the powder samples. Spot checks in a few cases showed less than 6% weight loss on holding 1hour at 105’ C. Sample 2 in the tables (furnished by Arnold Rosenberg, Dewey, and Almy) was prepared by a combination of ion exchange to remove cations and dialysis to remove materials of low molecular weight. Samples 3 and 4 (furnished by S. N. Yu, Dewey, and Amy) were purified by precipitation of a fraction from a concentrated solution with acidified methanol solution. Sample 1 was obtained from the Pulp and Paper Research Institute of Canada (through the courtesy of D. A. I. Goring) and was represented as a purified lignosulfonate. Samples 7 and 13 are sulfonated lignins obtained from the West Virginia Pulp and Paper Co. The remainder of samples are proc-

Reproducibility Runs

Direct spectra 280 mp 282 mp 200 209 208 210 197 207.5 196 205.5 12 6

4.5 2.2

of Ultraviolet Spectrograms of Lignosulfonates

Readings, millimeter@from zero lines Difference spectra A 340 mu A 300 mu A 250 mfi” A 250 mpb 31 64 153 141.5 146.5 144.0 30 61 152 143 31 61 31 62 152 142 1 3

3

5

7

5

3

2

A 229 mp

16 2 10 13 14

Very variable 0.056

Absorbance unite 0.048 0.018 0.004 0.012 0.028 0.012 a Gross reading from zero line established by solvents. Net reading from base line connecting minima a t 229 and 278 mp. 1 mm. = 0.0039 absorbance. Data at 280 and 282 mp are peak maxima of 0.1N acid and 0.1N alkali solutions of same lignosdfonate. All other data are at maxima or minima in difference Spectrogram. Concentration 100 p.p.m.; cell paths 1 cm.

’

214

ANALYTICAL CHEMISTRY

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was found to vary by a maximum of 2% in four successive runs each involving a separate dilution of stock solution over a 2 d a y period. The averages of two runs on each day by the base line method were 142.2 and 142.5 mm. or a difference of less than 3 parts per thousand. Fair agreement tcith Beer’s law is observed over the range 20 to 200 p.p.m. solutions of a lignosulfonate as shown by Table 11. Precision is improved by use of the base line procedure to estimate absorptivities. The base lines for the A250-mp maxim2 are obtained by drawing straight lines connecting the minima a t 229 and 278 mp. The base line for the A300 maximum is obtained by drawing a line connecting the A278 minimum with the intercept at A346 mp as shown in Figure 2. The distance from the base line to .;he peak maximum is estimated to within 0.5 mm. by a measurement with a ;straight-edge millimeter ruler. One millimeter corresponds to 0.0039 absorbance and therefore the uncertainty of a measurement is of the order cf 0.002 absorbance or about 3 parts per thousand at the 250 mp peak in the difference spectra data reported in Table 11. The standard deviation nithin the range 40 to 200 p.p.m. in an 0.5-cm. cell is 0.09 part on an average absorptivity of 7.06 by the base line method a t A250 mp in the difference spectnm, or 1.3%. The data in Tables I and I1 indicate that the ultraviolet spectrograms are reproducible to within *27G in yielding estimates of the lignosulfonate concentration in dilutions as low as 20 p.p.m. by our preferred method of measuring the difference spectrtm peak maximum

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at about A250 mp by the base line method, using 1-cm. cell paths. Levels as low as 1 to 2 p.p.m. are readily measured using 10-cm cell paths. Substantially greater sensitivity is achieved a t shorter wavelength (205 mp) ( l a ) ,but specificity is sacrificed and phenolic hydroxyl content cannot be

estimated because the absorption in this region is not specific t o the modifying influence of the aromatic hydroxyl group. QUANTATIVE EVALUATION OF SPECTROGRAMS

In Table I11 are presented ultraviolet difference spectra data for a number of guaiacyl derivatives considered to be possible models for the lignins and the lignosulfonates. Examples 1 and 2 are eugenol and conidendrin, which were used as model substances by Goldschmid (7). The other examples are based on the data of Aulin-Erdtman (3). Examples A,B,D, and E are plotted in relative absorbance along with a tracing of a softwood lignosulfonate in Figure 3. Examples F,G, and H are plotted in Figure 4,with tracings of the same softwood lignosulfonate as in Figure 3. Also shown is a tracing of a hardwood lignosulfonate. The difference spectrum of the hardwood type is significantly different from the softwood type. The hardwood-type stronger maximum is shifted toward longer wavelengths and the weaker maximum toward shorter wavelengths. All the individual plotted points are computed from Aulin-Erdtman’s data (3). These results are plotted in relative absorbance with the maximum absorbance normalized t o a value of 0.700 for comparison purposes. Spectrophotometric data on some technical lignosulfonate preparations

W A V E L E N G T H , m)J

Figure 3. Difference spectra of softwood lignosulfonate and s o m e reference substances Softwood lignosulfonate

- -4-Hydroxy-3-methoxytoluene-w-rulfonic acid, barium salt, A 8 - - - - - 4-Hydroxy-3-methoxybenzyl alcohol, 8 C)-4-Hydroxy-3-methoxy-l -propylbenzens, D e - . . . .- 6-Hydroxy-5-methoxy- 1 -methyl-3-propylbenzene, E 0

*

V O L 36,

NO. 1, JANUARY 1 9 6 4

215

are shown in Table IV for purposes of identification. A wide range of possible degrees of purity is observed. Difference spectrogram results for these preparations are presented in Table V. All absorptivity values in Table V were calculated from solvent zero absorbance tracings, except the values in column D , which were calculated from base line absorbance values. Per cent phenolic hydroxyl content as estimated by Goldschmid's method and the method suggested in this paper are tabulated in Table VI. These methods are:


Goldschmid's method for per cent phenolic hydroxyl is equal to the absorptivity of the long wavelength maximum a t about 300 mp in the difference spectrum, multiplied by 0.414. The method of this paper is equal to the corrected absorptivity of the short

Table It.

wavelength maximum a t about A 260 mp in the difference spectrogram multiplied by the factor 0.192. The corrected absorptivity is estimated from a base line connecting minima a t about A 229 and A 278 mp. This is Method B in Table VI. Also shown in Table VI is a computation of per cent hydrosyl obtained by multiplying the gross absorptivity at A250 mfi in the difference spectrum by 0.192. The results are 4% higher on the average for purified preparations (numbers 1 to 4) than the values obtained by the base line Nethod B. The gross absorptivities a t 4250 m,u appears to be less reliable data because of the lower precision achievable and because of possible interference due t o impurities which raises the base line in the difference spectrum. The l)ase line

Beer's l a w Tests of Ultraviolet Spectrograms of Lignosulfonates Using 0.5-cm. Matched Cells

hbsorptivity, l.g.-l cm.-l -Direct spectra Difference spectra _ _ _ _ Dilution, p,p.m. 280 mp 2S2 r n M A 250 n i p A 250 mpb A 300 mpa A 300 nipb 3.40 2.53 20 9.00 9.90 7.50 7.50 3.30 2.48 40 7.20 7.15 9.40 9.70 100 7.08 3.4s 2.58 9.77 7.26 9.40 3.1s 2.70 6.76 6.94 150 9.42 10.02 200 7.09 3.49 2.80 10.72 7.55 9.88 9.42 10.04 7.25 7.15 3.37 2.62 Av. absorptivity 0.21 0.13 0 12 0.31 0.41 0.31 Std. dev. Peak height measured from solvent zero line. * Peak height measured from base lines (Figure 2). Data at 280 and 282 mp are peak maxima of 0 . W acid and O.l.\' alkali solutions of 8nme lignosulfonate. ,411 other data are at maxima or minima in difference spectrogram.

Table 111.

Mode10 substance

Ultraviolet Difference Spectrophotometric Absorptivities and Phenolic Hydroxyl Ratios of Lignosulfonate Model Substances Ultraviolet difference spectra maxima Ratio of % hydroxyl Ratio to absorptivity Phenolic Short R-avelength Long wavelength of M p

Absorptivity 49.0 NDA 36.8 63.5 67.5 51.7 43.6 27.7 45.4 33.6

M/l 4 298 4 301 4 295 A 293 A 296 A 298 A 296 A 296 A 297 A 298

Absorptivities 24.8 24.6 13.7 25.7 25.7 25.8 27.0 19.7 16.4 15.3

A 247 NDAs A 4 250 B A 248 C A 250 D 4 247 E 4 249 F 4 257 G 4 254 H A 255 0.5 mole. Peak to peak distance in difference spectrum. Average 0.192 (range f15%). Average 0.402 (range 3=5%). No data available. Substance A. 4-Hydroxy-3-methoxytoluene-w-sulfonicacid, Ba salt B. 4-Hydroxy-3-methoxybenzyl alcohol C. 4-Hydroxy-3-methoxybenzyl alcohol D. PHydroxy-3-methoxy-I-propylbenaene 1

2

nbsorptivities 1.96

1 mpb

2.08 1.41 2.77 2.20

17 39 13

43

0

E. F. G. H.

6-Hydroxy-5-methoxy-1-methyl-3-propylbenzene

Dehydrodiisoeugenol

Dihydrodehydrodiisoeugenol S-[l-(Phydroxy-3-methoxyphenyl)]-propylthioglycolic

216

Method B corrects for nonspecific broad absorption in the difference spectrum. The factor 0.414 was computed from the average of Goldschmid's data for eugenol and conidendrin. The values entered in Table I11 for eugenol (example 1) are our own measured data. Goldschmid ('7) measured the absorptivities of these compomids a t about A300 mp, The per cent phenolic hydroxyl divided by the absorptivity yields the conversion factor for this method. The factor 0.192 was computed by averaging the ratio of the per cent phenolic hydroxyl to the absorptivity of the strong maxiniiim in the difference spectrum a t about A250 mp for substances 1 and iz, €3, D. and E listed in Table 111. This factor is believed to be a rcasonable figure for estimation of per cent hydroxyl from absorptivity values of the short wavelength peak in the difference spectrum of softwood lignosulfonates The validity of this figure depends on the validity of the models selected for compariwn. AI1 the models averaged are nonconjugated phenolic substances containing the guaiacyl nucleus with a saturated side chain para to the hydroxyl group. Base line data were not available for these models, with the exception of eugenol. The base line absorptivity of eugenol yieldpd a factor of 0.194 for ratio of per cent hydrosyl, which is close to the mean of 0.192for models 1 and A, B, D. and E. RIethosyl-phenolic hydroxyl molar ratios for some technical preparations are tabulated in Table VII.

acid, Na salt


Solvent Water Water

Alcoh 01 Alcohol .4lcohol

Alcohol Alcohol Water

Molecular weight

hydroxyl,

1 A4

10.39 9.54 5.92 11.02 11.02 10.24 9.45 5.20 5.16 9.52

1 SO 327 329 27s

%

Short wavelength 0,2126 KDA 0.161' 0 . 174c 0.163 0.199c 0.217. 0.187 0.113 0.282

Long wavelength 0.41gd 0 . 388d 0 . S32d 0.429d 0 42gd 0.397 0.349 0.263 0.313 0.621 I

Table IV.

darnple

1 2 3 -I

5 (3

7

s 9

10 11 12 13 14 15

16 17


1s

Ultraviolet Spectrophotometric Data for Some Crude and Partly Purified Lignosulfonates

Ratio of 282-mp Direct spectra absorptivities and 205 280-mp Identity Type 280 mp 260 mu 2S2 m p mp peaks 1,090 9.40 14.24 SC 13.04 Purified LSb 15.20 1 .08*5 10.98 Dialyzed LS S 14.00 1.073 9.28 13 54 Solvent fractionated LS S 12.60 1.072 S . 95 13.70 12.80 Solvent fractionated LS S 1.095 6.60 10.40 S 9.49 LS 1.052 7.35 10.30 S 9.7s LS 13.98 66 1.110 S 12.60 11.60 Sulfonated ligoin 10.30 1.026 S 10.06 6.64 LS 1.084 7.75 10.10 S 9.30 L8 1,018 7.23 10.10 S 9.92 LS 8.32 67 1.090 S 7.65 5.15 LS 1.068 7.05 10.80 LS S 10.10 66 1.10 11.70 13.70 Sulfonated 1ig:iin S 12.40 5.90 64 1.087 s 8.20 6.40 Ld 9.74 63 1.093 S 8.90 7.46 LS 1,s 5.89 S.12 1.190 ? 6.83 LS ? 7.70 6.58 8.85 1.150 LP S 5.30 3.70 5.50 1.036 ~

In 0.1N potassium hydroxide. Other wavelengths are for 0.lX HC1 solutions. Ijata for 205 mp are for 100 p.p.m. concentrations in 1-mm. cells. Other data are for 100 p.p.m., 10-mm. cells. Lisnosulfonate. c Softwood.

1NTERPRETATlON OF DIRECT ULTRAVIOLET SPECTROGRAMS OF LIGNOSULFONATES

The absorptivities of purified softwood lignosulfonates a t 280 mp in neutral or acidified sclution range from 13 to perhaps as higi as 17 (IO, 15), which is comparable in magnitude to values listed in Table VI11 for simple model substances such as eugenol. The introduction of either a hydroxyl or methoxyl substituent into the benzene nucleus (phenol and anisole) results in a displacement of the secondary maximum of benzene from 254 inp to the 270- to 280-mp region accompanied by a five- to sixfold increase in intensity (6). The introduction of a second substituent of the other kind in tl-e ortho position (guaiacol) results in only small further changes in intensity or displacement of the band. Substitution of a nonionizable, nonconjugated grouping in the para position of guaiacol causes the peak maximum to center a t about 280 mp and the absorptivity reaches a steady value of about 18 to 20 in the models considered. Sonconjugated para-substituted guaiacyl model substances such as eugenol or conidendrin display a fairly constant ratio of per cent niethoxyl to absorptivity ranging from 0.92 to 1 1 whirh encompasses the values listed for purified lignosulfonates and for native lignin. Processed lignosulfonates average somewhat lower in ratio of per cent niethoxyl to absorptivity at 280 mp. and the spread of values is large, ranging from 0.70 to 1.28 in a group of commercial lignosulfonate retarders (9). Even lower ratios are observed for kraft

lignin and sulfonated lignin. Lower values of the ratio would indicate the presence of other chromophores which absorb more intensely, such as conjugated carbonyl ( I d ) and diphenyl (IS). Another possible cause of a lorn ratio is loss of methoxyl groups by degradation during the cooking process I

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or in subsequent treatment. A third possibility is the presence of impurities such as furfural which absorb strongly a t 280 mp ( l a ) . The wide variations observed would severely limit the value of the direct absorption a t 280 mp for quantitative applications. The much lower ratio of per cent hydroxyl to absorptivity in the lignins compared to the suggested models is not unanticipated. Most of the absorption is due t o methoxyl groups. In alkaline solution the secondary or B band of softrrood lignosulfonates undergoes the following changes from the neutral form which is associated with ionization t o the phenoside ion: Broadening of the band. Displacement of the maximum from 280 t o about 282 to 283 mp. Increases in intensity of about 7 to 10%. The question is raised whether the order of magnitude of the observed changes is consistent with phenoside ion content in alkaline solution of lignosulfonates. The direct spectrum of the neutral form of a simple phenolic substance (unhindered, and with no other ionizable groups or highly polar substituents on the ring) may be considered to consist of a secondary band a t about 260 to 280 mp and of a primary band or shoulder a t about 235 mp. Ionization of the hydroxyl group results in shifts of these bands toward longer wavelengths, accompanied by increases in intensity. Conjugation of the phenI

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050-

040-

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030-

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280

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270

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320

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WAVELENGTH, rnu ,

Figure 4.

. -- ---

- e _ . _

000-

Difference spectra of lignosulfonates and some reference substances Softwood lignosulfonate -Hardwood lignosulfonate * -Dehydrodiisoeugenol, F Dihydrodehydrodiisoeugenol, G S- [(1-(4-hydroxy-3-rnethoxyphenyl)] -propyl thioglycolic acid, rodlum salt, H

9

. - .- . -

VOL 36, NO. 1, JANUARY 1964

217

Table V.

Ultraviolet Difference Spectra Absorptivity Data for 1 8 Representative Lignosulfonates"

A

A

Sample I

2 3 4 5

6 7 8 9

10

11 _12 _


13 14 15 16 17 18

B

D

C

F

E

Difference spectra absorptivities 340 mu A 300 mp A 250 mp A250 mp (gross) (gross) (gross) (base line) 2.20 4.20 11.00 10.42 2.60 3.80 10.40 10.30 1.34 3.95 10.85 10.2 3.20 1.33 9.56 9.78 1.23 4.28 9.53 10.30 1.74 8.83 3.69 9.15 3.66 8.74 5.62 10.65 1.65 3.30 8.21 8.48 1.19 8.01 7.93 3.90 0.96 7.99 3.14 7.61 . . ~ 1.40 7.55 3.G 7.86 1.13 7.55 3.24 8.40 3.40 4.94 7.03 9.06 1.20 6.60 3.25 7.07 1.55 6.43 3.29 6.90 2.50 1.00 4.55 5.50 1.20 2.40 6.17 5. i2 0.82 2.25 5.00 5.25

Difference (C D) +0.58

-

$0.10

+0.65 +0.22 $0.77 $0.22 $1.91 $0.27 $0.08 $0.38 $0.31 $0.85 +2.03 +0.47 $0.47 -0.95 +1.05 +0.25

Difference spectrum, strong peak maxlmum, mp 251.0 250.5 251.0 251.3 250.5 250.0 250.0 251.0 250.5 250.0 250.5 250.5 250.2 250.5 251.5 252.5 252.0 250.8

G

mP 48.5 49.5 48.0 48.7 49.0 49.0 51.0 49.5 49.5 A

50.0

49.5 49.6 51.0 49.5 48.0 44.5 46.0 49.4

H

i

Absorptivity ratios D/B D/A 2.48 4.74 2.71 3.96 2.59 7.60 2.99 7.25 7.75 2.23 2.40 5.08 1.55 2.39 2.49 4.96 2.06 6.74 2.43 7.93 2.39 5.40 2.33 6.68 1.47 2.06 5.49 2.04 1.95 4.14 2.20 5.50 2.13 4.25 2.22 6.10

a Conditions. 0. I N potassium hydroxide solution of lignosulfonate in sample beam of Beckman DK-2 recording ultraviolet spectrophotometer. 0.1N HC1 solution of identical quantity in reference beam. Concentrations 100 p.p.m. Cell paths 0.5 or 1.0 cm.

polar) can be estimated approximately Table VI.

Sample 1 2 3

4

10 11 12

13 14 15

16 17 18 a

%

Phenolic Hydroxyl in Lignosulfonates Listed in Tables IV and Estimated by Ultraviolet Difference Spectrum Method

Phenolic hydroxyl, % ' Goldschmid's This paper Method Aa Method Bd 1.74 2.00 1.57 1.98 1.63 1.96 1.32 1.85

1.30 1.30 1.34 2.08 1.34 1.36 1.03 1.00 0.93

1.46 1.45 1.45 1.35 1.29 1.26 1.05 0.98 0.96

This paper Method Cc 2.12 2.00 2.1s

1.53 1.51 1.62 1.74 1.36 1.32 0.87 i.ig 1.01

V

Ratio (AD). Goldschmid's/ this paper 0.87 0.79 0.82

0.89 0.90

0.92 1.54 1.04 1.08 0.98 1.02 0.97

A. Column B of Table V times 0.414 (Goldschmid's method). Column D of Table V times 0.192 (base hne method). C. Column C of Table V times 0.192 (gross absorbance method).

* B.

oxide ion with the ring offers additional opportunities for aromatic resonance, which favors a still stronger absorption shifted even further toward longer wavelengths (6). The data in Table IX show that the increase in intensity at 280 mp of phenol, guaiacol, eugenol, and purified lignosulfonates due t o ionization in alkaline solution is proportional to the hydroxyl content, since a fairly constant ratio of per cent increase t o per cent hydroxyl is observed, the range being 4.1 t o 5.1 The magnitude of the displacement in

218


wavelength in the examples in Table IX which contain the guaiacyl structure is roughly inversely proportional t o the ratio of ether linkages to hydroxyl in the molecule. Methoxyl groups tend to stabilize the absorption at 280 mp and the shift will decrease as the proportion of hydroxyl to methoxyl and other ether groups decreases. The per cent phenolic hydroxyl in nonconjugated phenols with at least one ortho position occupied by hydrogen and with no other ionizable or strongly polar substituents (methoxyl and alkyl substituents are moderately or weakly

as follows: Per cent phenolic hydroxyl = 0.21 X per cent increase in intensity of secondary maximum in the 270- to 280-mg region in alkaline solution compared with neutral solution

A phenolic hydroxyl content of 2% in a purified lignosulfonate corresponds to about 0.25 phenolic hydroxyl unit per unit weight of 220. In a purified lignosulfonate with an aromatic methoxyl content of 12.8%, a level of 2% phenolic hydroxyl corresponds to a molar ratio of 3.5 methoxyls to 1 phenolic hydroxyl. The above equation applies only to

Table VII. Methoxyl-Phenolic Hydroxyl Ratios of Some Crude Lignosulfonates

Molar ratio of

methPer cent oxyl Phenolic to SamMeth- hyhyple Sulfur oxyP droxylb droxyl 7 9.5 1.67 1.97 6.0 6.75 1.29 2.63 14 6.2 7.4 1.26 3.22 15 3.9 1.26 2.39 5.5 19 3.9 1.35 2.60 20 6.4 7.2 21 1.32 2.98 1.19 3.36 22 7.3 23 7.5 1.30 3.22 1.19 3.46 24 7.5 25 7.1 1.30 3.00 26 7.3 1.27 3.16 27 8.2 1.19 3.76 3.64 28 8.2 1.23 By Zeissl micromethod. * By base line method of this paper.

Table VIII.

Absorptivities of Lignosulfonates Compared to Model Substances Location of Ratio of Ratio of secondary Absorptivity % CHaO t o 70 OH to Molecular absorpabsorp 0: Substance Reference weight % Methoxyl % ' Hydroxyl maximum, mp maximum tivity tivity D Benzene 78 254 2.6 Phenol 95.6 17.8 269.5 16.4 1.02 Anisole 108.13 28.6 270 13.7 2.09 e 1.41 Guaiacol 124.13 24.9 13.7 274 17.6 0.78 a 1.11 Eugenol 164.1 18.9 10.36 17.0 0.61 0.57 156.1 20.0 10.87 281 19 1.06 Vanillyl alcohol (14) Dihydro eugenol 166.1 18.7 10.22 18.1 1.03 0.57 Conidendrin (') 178('/t) 17.4 9.6 284 18.9 0.92 0.51 Dihydrodehydro diisoeugenol ('(3) ) 297 18.9 11.4 282 20.2 0.56 0.93 14.8' 3.87b 280 16.2 0.92 0.24 Native lignin (hemlock.) (8) Pine kraft lignin 0.15 13.gb 3.13b 280 21 0.66 12.8 2.0 280 14 0.14 0.92 Purified lignosulfonate (18) 11.6-11.7 1.5 280 10.50 1.11 0.14 (11) Sulfonated lignin 0.13 6.0 1.7 280 12.8 0.47 13 Commercial lignosul0.84 0.15 6-8 1.3 280 7.8-11.8 fonates (9) a 9 Sam les of lignosulfcnates 0.15 0.86 7-8 1.3 280 8.5-8.7 0.44 5.4 278 12.36 Kraft! nin (9) 0.70 8.2 11.78 LignosuYfonatea, NH, t alt (9) (9) Lignosulfonate, calcium salt 1.28 11.8 9.18 (I


(I

a

This paper. (7).

softwood lignosulfonsttes of relatively high purity. It will f a d to hold for sulfonated lignins, kraft lignins, hardwood lignosulfonates, and degraded or low purity material. TEe difference spectrum is a more reliable means of estimating phenolic hydroxyl content of low purity softwood lignclsulfonates. Summing up, the direct spectrum of purified softwood ILgnosulfonates is comparable to the d rect spectrum of certain model substances containing nonconjugated guaiac:yl units in the following respect: Ratio of per cent methoxyl to absorptivity a t 280 mp is 0 9 to 1.1, which is comparable to 1.11 For eugenol, 0.92 for conidendrin, and D.93 for dihydrodehydrodiisoeugenol. The per cent increase in the secondary maximum at about 280 mp due to phenoxide ion formation is proportional to the phenolic hydroxyl content. The peak displace nent in alkaline solution is of the order of magnitude anticipated for repeat units containing a ratio of about 1 guaiacyl unit to 4 etherified rings. INTERPRETATION OF 'THE ULTRAVIOLET DIFFERENCE SPECTRA OF LIGNOSULFONATES

Phenol - Phenolal e Comparison. The ultraviolet difference spectra of the model substances in Table I11 may be placed in two groups based on the position of the strong peak and t o some extent on the Amp value or the spacing between the !strong and weak peak maxima in millimicrons. Group 1 consists of simple, nonconjugated guaiacyl units without branching a t the carbon in the side cEain para to the hydroxyl group. These substances exhibit strong peaks n the difference

spectra R-ith maxima in the range A247 to A250 microns. The Amp values for this group range from 45 to 51 microns with an average of about 47.8 microns. Substances 1, A, B, C, D, and E are in this group. I n the second group are nonconjugated guaiacyl derivatives with branching a t the carbon. Substances F, G, and H belong to this group. These have the strong peak maximum displaced toward longer wavelengths between A254 and A257 mp. They show lower Amp, values ranging from 39 to 43 mp. Insufficient data are available a t this time about the branched substance conidendrin to place it in either group. All these nonconjugated model substances fail t o absorb appreciably above A320 mp, in sharp contrast to conjugated guaiacyl derivatives such as vanillin and isoeugenol with an a-carbony1 and an aethylenic group, respectively. The major effect of conjugation of the aromatic hydroxyl with a carbonyl group in the para position is a displacement of the long wavelength

difference spectrum peak by some 50 to 60 mp, accompanied by large increases in intensity. Little displacement of the short wavelength peak is observed in these two examples. In contrast, isoeugenol displays a strong negative absorption at A250 mp and a very broad band with two peaks in the A280- to 340-mp region. The difference spectra of phenolic substances may reveal negative absorption in regions where the neutral or acidified forms absorb more strongly than the ionized or phenolate form as in the cases of eugenol and dihydroeugenol a t about A280 mp or of vanillin and syringealdehyde a t A230 and A300 mp. The unbranched species (1, A to E of Table 111) show remarkably constant ratios of per cent hydroxyl to A300 mp absorptivities ranging from 0.395 to 0.432 and therefore this maximum should be suitable for estimation of molecular weights of similar species from difference spectra data within *5QI,. The corresponding ratio for the strong peak maximum is more sensitive to the

Table IX.

Changes in Secondary Maximum in the Ultraviolet Due to Ionization of Phenolic Hydroxyl in Alkaline Solution Ratio Ratio of of yo ether Peak Phenolic increase linkages shift, Increase, hydroxyl, to % to Substance yo hydroxyl hydroxyl mp % 1. Phenol 91 17.8 5.1 ... 2. Guaiacol 15 l8 64 13.7 4.7 1 3. Dihydrodehydrodiisoeugenol 5" 21.6" 5.18 4.2 3 4. Eugenol 16 49 10.4 4.7 1 5. Purified lignosulfonateb 2 8.0 1.95 4.1 5-6 e Computed from data in ( 4 ) * Average of softwood examples 1 to 4 in Table IV and VI. ~~

~~

VOL. 36, NO. 1, JANUARY 1964

~~~

219

,

00

J

I

I

I

I

I

I

I

I

I

I

I

1


0 70

WAVELENGTH, my

Figure 5.

Difference spectra of lignosulfonate and lignosulfonate plus vanillin

.__ 100_ p.p.rn. _lignosulfonate 100 p . p m lignosulfonote plus 1 p , p m vanillin

type of substituent in the position para to the hydroxyl group, but the range is still relatively narrow with a spread from 0.161 to 0.212 for substances 1 and A, B, C, D, and E. Therefore, the phenolic hydroxyl content of similar species can be estimated within *15% by measurement of the absorptivit>-of the strong peak in the difference bliwtrum a t A250 mp. The difference spectrum strong peak of a softwood lignosulfonate shown in Figure 3 matches closely with model substance A in this region and fairly well with the group, 1, A, B, C, D, E. The weak peak of either the softwoodorhardwood lignosulfonate does not match well with any of the nonconjugated substances discussed so far. The lignosulfonates absorb significantly at and above A320 mp and this absorption appears to overlap with the weak peak a t about A300 mp (Figures 3 and 4). ilbsorption in this region has been attributed to the presence of conjugated elements (17). The absence of a maximum at about A350 to 360 mp would tend to rule out significant amounts of carbonyl conjugation as exemplified by vanillin or syringealdehyde. The possibility of overlapping absorption a t about A300 mp in the difference spectra of lignins and lignosulfonates makes it difficult to draw meaningful inferences and conclusions about the phenolic hydroxyl content of these substances by comparison with the “clean” weak peak in the model substances considered in Table 111. The stronger peak a t A250 mfi seems to be a better choice for comparison. The

220


possibility of overlap of spectral bands due to other species of absorbing elements can be partly compensated by using the base line technique suggested in this paper for the estimation of absorptivities. The presence of vanillin and syringealdehyde in incompletely purified specimens is readily detected by measuring the incremental absorption in the A350 to 360-mp region and applying a correction. The difference spectrum strong peak niaxima of the lignosulfonates listed in Table V fall within a narrow range of A250 to 251 mp) with the exception of samples 16 and 17 which are shifted toward longer wavelengths. The A m p value or spacing between the strong peak and the weaker peak (near A300 mp) falls within the range of 48 to 51 mp, which appears typical of softwood type lignosulfonates. Examples 16 and 17 again are exceptional in exhibiting significantly smaller Amp values. Comparison with a hardm-ood lignosulfonate (Figure 4) is suggestive of a hardwood source for samples 16 and 17. The absorptivities of the strong peak in the difference spectra have been computed in two ways. In column D in Table V is the absorptivity computed by the preferred base line method. In column C is the absorptivity computed from zero lines established by tracings of solvents in the same cells used for solutions of samples. The base line values are usually smaller by 5 to 10% in softwood lignosulfonates. The larger differences in the examples of sulfonated lignins (7 and 13, Table V) may be attributed to their anomalously high base

lines, which indicated a somewhat different pattern of absorption. The ratio of the base line absorptivity of the strong peak maximum to the absorptivity a t A340 mp (region of strong absorption in conjugated phenols) falls within a range of about 4 to 8 for most examples, with two significant excep tions, sulfonated lignin examples 7 and 13. These have anomaIously high absorptivities at A340 mp of 3.66 and 3.40, compared n-ith an average of 1.80 for examples 1 to 6 in Table V, The evidence of the spectrograms suggests overlapping of the longer wavelength absorption with the A300 mp-peak. The result is an excessively high absorptivity by as much as 2.0. The stronger peak a t about A250 mp would appear a better choice for estimation of the phenolic hydroq-1 content, provided comparisons with model substances are made by the base line method. The much better fit of the difference spectrum strong peak with model substance A would indicate that this peak is a better choice than the A300-mp peak for analysis and comparison of the lignosulfonates. The interference of vanillin, syringealdehyde, and similar conjugated phenols is not a problem in purified materials. Their presence can be detected by appearance of a peak a t A350 to 360 mp, as shovin by Figure 5. The presence of lY0 vanillin causes a definite peak to appear at 350 mp with an increase in absorptivity from 1.5 to 3.6. The absorptivity of the difference spectrum strong peak has been raised from 6.5 to 7.3 in this example. The A300-mp peak has undergone a decrease due to the presence of vanillin. The absorptivity of vanillin is 171 a t 348.0 mp and 115 a t A248 mp in the difference spectrum. An excessive absorption of 1.8 a t A348 mp corresponds to an excess of about 1.1 a t A250 mp, which agrees well Kith the observed increase a t A250 mp in the mixture of 100 p.p.m. of lignosulfonate (not purified) and 1 p . p m of Tanillin. It is possible to remove the interference due to vanillin, syringealdehyde. and other small molecules by extracting a slightly acidified solution of lignosulfonate with ethyl ether. These results indicate that the absorption of conjugated elements of the vanillin type a t A250 mp is one half the A350-mp absorption. This amounts a t most to about 10% of the total absorption. The effect of this mould be to make the apparent per cent hydroxyl content too high by 0.1% in purified preparations and 0.05% in commercial preparations. This is evident from a comparison of the absorptivities of conjugated phenols (about 100) with nonconjugated phenols (about 50) a t A250 mp. Thus, example 1 in Table V would have an excess absorptivity a t A250 mp of 2.2/2 = 1.1.

The conversion factor to per cent phenol is 0.2 in nonconjugated elements and 0.1 in conjugated elements a t A250 mg. It is the difference between these tn-o values which contributes an excessive per cent hydroxyl value or a value of 0.1% in the phenolic hydroxyl content attributable to conjugated elements. Commercial prepamtiom such as examples 9 to 12 average about 0.05 t o 0.06% phenolic hydroxyl content possibly attributed to conjugated elements. CONCLUEIONS


The direct ultraviolet spectrograms of softwood 1ignosLlfonates can be interpreted as conEistent with the guaiacyl hypotheses with a ratio of about 4 methosyls i o 1 hydroxyl on aromatic rings. The lifference spectrograms are also consistent with the guaiacyl hypothesis. The most useful

datum of the difference spectrogram is the base line absorptivity a t 250 mp for estimation of aromatic hydroxyl content. ACKNOWLEDGMENT

The assistance of Frank D. Brako of Dewey and Almy in obtaining many of the spectrograms is gratefully acknowledged. LITERATURE CITED

69. 3780-3- I19.53). I

\ - - - -

(9)-Halstead, W. j:,Chaiken, B., Public Roads 31, 126-35 (1961). (10) Harris, E. E.. Hogan, D.. I n d . Ena. ' Chem. 49: 1393 i195fi. ' ' (11) Jensen, W., Fieme;, K. E., Forss, K., T a p p i 45, 122-7 (1962). (12) Kleinert, T. N., Joyce, C. S., I b i d . ,

40, 813-21 (1957). (13) Kolboe, S., Ellefsen, O., Ibid., 45, 163-6 (1962). (14) Lemon, H. W., J. Am. Chem. SOC. 69, 2998-3000 (1947). (15) Moacanen, J., Felicetta, 5'. F.,

Haller, W., McCarthy, J. L., Ibid., 77,

(I) Aulin-Erdtman, G., S v e n s k - P a p p e r s t i d . 55,745-9 (1952). (2) Zbid., 56,287-91 (1953). (3) Ibid., 57, 745-60 (1954). (4) Aulin-Erdtman, G., Tappi 32, 160-6 (1949). (5) Butler, J. P., Czeipiel, I. P., ASAL. CHEM.28, 1468-72 (1956). (6) . . Doub, L., T'andenbelt, J. M., J . Am. Chem. SOC.69, 2714-23 (19-17). (7) Goldschmid, O., Isar,. CHEM.26, 1421-3 (1954). \ - -

(S) Goldschmid, O., J. Am. Chcm. Soc.

- - I

3470-5 (1955). (16) Patterson. R. F.. Hibbert. H.. Ibid.. ' 65,1862-9 (1943). ' (17) Zbid., pp. 1869-73. I

,

(1s) Ritter, D. M.,Olleman, E. D.,

Pennington, D. E., lT7right, IC. A , I b i d . , 72, 1347-51 (1950). (19) Sarkanen, K., Schuerch, C., ASAL. CHEM.27, 1245-50 (1955). RECEIVED for review January .4ccepted October 7, 1963.

14, 1963.

Spectrophotometric Determination of Dithiocarbamate Residues on Food Crops THOMAS E. CULLEN Niagara Chemical Division, FMC Corp., Middleport, N. Y. ,The standard carbon disulfide evolution technique for determining dithiocarbamate residues on food crops has been revised. The residue is decomposed on the crop and the evolved carbon disulfide is collected and reacted to form the yellow cupric salt of N,N-bis(2-hydroxyethyl) dithiocarbamic acid which can be measured colorimetrically. The speed of decomposition of the dithiocarbamate and the copper-carbon clisulfide ratio are found to be critical factors. An improved reagent is presented tooptimize the effect of these factors. The procedure presented ha:; proven to be applicable to most dithiocarbamates. Recoveries of 85 to 100% of theoretical were generally obtained on all dithiocarbamates for every crop tested. The method is sensitive to 20 p g . of carbon disulfide.

T

of dithiocarbamic acid as fungicides against a variety of plant pathogenic fungi has necessitated the development of an accurate analytical method for their detection and determination on food crops. The colorimetric method for the determination of dithiocrtrbamate residues was first published Ey Clark ( 4 ) and Lowen (IO). The method was based

upon earlier work by Viles (16) and Dickenson (5). Pease (12) and Hauermann (9) later proposed the technique which has been generally accepted. This method is based upon the acid decomposition of dithiocarbamates to carbon disulfide and the corresponding amine. Alkyl monodithiocarbamates can decompose only to these products. However, as has been previously observed ( 4 ) , the decomposition of bis(dithiocarbamates) can also yield hydrogen sulfide. The carbon disulfide which evolves from the decomposition reactions is absorbed in an ethanol solution containing cupric acetate and an alkylamine to form the yellow chelates ('7, 8, IS): CS2

+ RINH + CU"

HE USE OF DERIVATIVES

2CS2

-t

+ 2RzNH + Cut'

-*

EXPERIMENTAL

(1,l-complex)

R

\

RN ,s\-";.',.

mining copper (2, 3, I 7 ) , tetramethylthiuram disulfide ( I ) , and amines ( I I , l ~ as ) well as carbon disulfide. -4 general disagreement concerning the exact wavelength that should be employed for measurement of cupric diethyldithiocarbamate has been noted. Various workers have used wavelengths of 380 mp ( l a ) , 400 mp ( 2 ) , 425 mp (3, 4) and 435 mp (9). This disagreement is discussed in later sections of this paper. Purified samples of these typical commerical fungicides were chosen as standards: zineb, [ethylene-bis (dithiocarbamato) ] zinc; maneb, [ethylene-bis (dithiocarbamato) ] manganese; ziram, di-(N,.V-dimethyldithiocarbamato)zinc; ferbam, tri-(X,N-dimethyldithiocarbamato) iron; thiram, bis(N,N-dimethylthionocarbamyl) disulfide; metiram, a mixture of [ethylene-bis(dit1iiocarbamato) ] zinc and [dithiobis (thiocarbonyl)iminoethylene]bis (dithiocarbamato)zinc.

AA

R

/ R'

(1,2-complex) The formation of these chelates has been the basis of methods for deter-

Apparatus. The apparatus used to decompose the dithiocarbamate residue and collect the carbon disulfide is illustrated in Figure 1. This is an adaptation of an apparatus described by Clark (4). The addition funnel is equipped with a tip long enough to reach within 1 cm. of the bottom of the reaction flask so as to provide agiVOL. 36, NO. 1 , JANUARY 1964

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221

Characterization of Lignosulfonates by Ultraviolet Spectrometry. Direct

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