ON THE USE OF ZnCl 2 TOGETHER WITH HCI IN LIGNIN DETERMINATIONS L. Paloheimo and K. A. Vainio Department of Animal Husbandry, University of Helsinki Received October 21, 1958. As early as 1852 Barreswil (ref. Stamm 5, p. 160) found that hot concentrated zinc chloride solution swells and dissolves paper. The first to recommend the use of ZnCl 2 in lignin determination was J. König (1, p. 65). König claimed that a mixture of 40 g Zncl2 and 100 ml concentrated hydrochloric acid was a convenient hydrolysator to be used in lignin determinations. As the formation of HCI gas is very intense he suggested the use of effective cooling. However, with the addition of 5 ml water to the mixture a temperature of 50° C should be appropriate. König, however, did not present any analytical data obtained from the use of this principle. In 1938 Poppoff (2, p. 245) published a treatise on the use of König' s principle in lignin determination. His mixture was: 40 g ZnCl.,, 100 ml concentrated hydro- chloric acid, and s—lo ml water. 20—30 ml of the mixture was used for 0.5— 1 g air dty matter to be analysed, the recommended temperature being 25° C, and the duration of the treatment 10 h. A 2 hours pretreatment with 2.5 per cent hydroch- loric acid was used. Protein correction was omitted. Experimental In our preliminary experiments filter paper was used as test material. It appeared that water solutions of ZnCl2 as well as solutions of ZnCl2 in diluted HCI decomposed the paper very slowly. The use of mixtures of ZnCl 2 and concentrated (37 p.ct.) hydrochloric acid caused a more rapid decomposition of cellulose. A mixture of 150 g ZnCl 2 and 100 ml 37 per cent. HCI appeared to be most efficient. In the following this mixture is called the V-solution. At 20° the V-solution dissolves 1 g filter paper in less than 1 hour, and the solution stays clear after dilution with water. Using the V-solution as hydrolysator the lignin determinations were made in the following way. 46 Table 1. Comparison of results obtained by the 72 per cent H 2S0 4 method and the V-method (ZnCl2 in HCI). Percentages on the dry matter basis. 72 per cent H 2 S04 method V-method Crude lignin 1 ) I Lignin Crude Lignin1 ) Lignin Spruce wood 23.4 23.2 Rye straw 13.2 12.7 13.1 12(5 Timothy S.l 7.6 8.5 8.0 2 g samples of the matter to be analysed were used. After a pepsin digestion the material was treated with boiling 2 n. hydrochloric acid, and then extracted with ethanol-benzene. The details of the pretreatments are described by Salo (3, p. 185). The residue is transferred into an 100 ml Erlenmeyer flask which is provided with a glass stopper. 50 ml V-solution is added in two portions. After the first addition the material is stirred thoroughly with a glass rod. The flask is put in a 20° water bath and is gently shaken at 15—30 minutes intervals. Towards the end of the treatment it can be shaken more vigorously. After 3 hours the contents of the flask are transferred into a 600 ml beaker and diluted with water up to a volume of 350 ml. The solution is heated to boiling point and then filtered according to the indication of Salo (1.c.). A protein correction is made. If woods are to be analysed the pepsin digestion as well as the protein correction are omitted. For the sake of comparison some determinations were made both with the method described above and by using 72 per cent H 2S0 4 in the way the latter method is used in our laboratory (described by Salo 1.c.). In both cases the pepsin digestion was applied. Table 1 shows that the two methods give, at least as regards the test materials used, highly uniform results. The lignin preparations received by the V-method are appreciably lighter in colour than those from the H 2S0 4 treatment. It is noticeable that the difference in colour does not reflect in the percentages. Table 2. Comparison of results obtained from the use of Konig’s solution with those obtained from the use of V-solution. Dilution after hydrolysis was omitted. Percentages on the dry matter basis. Konig's solution V-solution Crude lignin Lignin Crude lignin Lignin i Spruce wood 24.2 21.8 Birch Wood 14.» 9.4 Rye straw 12.6 12.1 11.0 10.6 Timothy hay 9.4 9.0 7.0 7.1 Red clover hay 7.1 6.3 6.S 6.1 ') Without protein correction. 47 Table 3. Influence bf the duration of treatment with the V-solution. Dilution after hydrolysis omitted. Percentages on the dry matter basis. Duration of Spruce wood Rye straw Timothy hay , Residue Residue Residue Residue Residue without without with without with protein protein protein protein protein correction correction correction correction correction 1 22.5 11.(1 10.5 9.8 9.3 2 21.6 I<>.7 10.3 8.0 7.5 3 21.8 11.0 10.6 7.6 7.1 5 21.2 11.0 10.6 8.0 7.5 7 21.0 11.2 10.8 7.6 7.1 Poppoff (1.c.) did not dilute with water after the treatment with the mixture of ZnCl 2 and HCI. For the sake of comparison we made some determinations with the V-method omitting the dilution. In Table 2 the results are compared with those obtained with the use of Konig’s solution (including 5 ml water). The pretreatments were in both cases similar to those described above. The duration of the hydrolysis was 3 hours, and the temperature 20° C. It should be noted that the terms »Crude lignin» and »Lignin» in Table 2 have not the same meaning as in Table, 1 because the figures in Table 2 are from a treatment without dilution with water. Table 2 shows that the residues received from the use of the V-solution are smaller than those with Konig's solution. It is not, however, possible to decide whether the greater efficiency of the V-solution is directed only to the cell wall carbohydrates or perhaps also to the lignin to a greater extent than the efficiency of the Konig’s solution. However, our experiments with filter paper have shown the superiority of the V-solution as a hydrolysator of cellulose. As for the behaviour of lignin in acid solutions which are used for decomposition of cell wall carbohydrates we refer to a treatise of Salo (1, p. 206). The hydrolysis time was fixed at 3 hours after a series of experiments the results of which are shown in Table 3. The materials were pretreated as before, and the dilution after the treatment with the V-solution was omitted. If the proba- bility of experimental errors is taken into account one may conclude that for rye straw and timothy hay a hydrolysis time of 1 hour should be convenient. For spruce wood a longer time seems to be needed. To allow a margin of safety a period of 3 hours was fixed. S u m mary As Konig’s proposal on the use of ZnCl2 together with hydrochloric acid in lignin determinations seems, after Poppoffs experiments, to have been neglected some experiments based on this principle were performed. A mixture of 150 g ZnCl 2 and 100 ml concentrated hydrochloric acid appeared to be the most effective for the decomposition of the cell wall carbohydrates. The results obtained were 48 highly comparable to those obtained from the use of 72 per cent H2 SO, as hydroly- sator. The lignin preparations were lighter in colour than those obtained by the H 2 S0 4 method. REFERENCES 1) König, J. 1930. Neues Verfahren zur chemischen Untersuchung der Futter- und Nahrungsmittel Berlin. 2) Poppoff, I. D. 1938. Zur Methodik der quantitative!! Ligninbestimmung. Z. Tierern, u. Futter- mittelkunde 1. 245—249. 3) Salo, M.-L. 1957. Lignin studies. I. Investigations concerning lignin determination. J. Sci. Agric. Soc. of Finland 29, 185—193. 4) —*— 1957. Lignin studies. 11. The lignin content and properties of lignin in different materials. Ibid. 29, 202—210. 5) Stamm, Alfred J. 1946, Cellulose solvents and the properties of cellulose in solution. Wood Che- mistry, edited by Louis E. Wise. New York. SELOSTUS: SINKKIKLORIDIN KÄYTÖSTÄ YHDESSÄ KLOORIVETYHAPON KANSSA LIGNIINIMÄÄRITYKSISSÄ L. Paloheimo ja K. A. Vainio Yliopiston kotieläintieteen laitos, Helsinki. Königin ehdottama prinsiippi sinkkikloridin käytöstä yhdessä konsentroidun kloorivetyhapon kanssa ligniinimäärityksissä näyttää sitten vuoden 1938, jolloin Poppoff julkaisi eräitä tätä prinsiip- piä noudattaen saatuja tutkimustuloksia, jääneen kokonaan unohduksiin. Periaatteen käyttökelpoi- suutta on nyttemmin kokeiltu kotieläintieteen laitoksessa, Soluseinämähiilihydraattien hydrolysaat- torina osoittautui tehokkaimmaksi seos, joka oli kokoonpantu 150 grammasta sinkkikloridia ja 100 millilitrasta väkevää (37 %) kloorivetyhappoa. Saadut tulokset ovat varsin yhtäpitäviä niiden tulos- ten kanssa, joita saatiin käyttämällä 72-prosenttista rikkihappoa hydrolysaattorina. Saadut ligniini- preparaatit olivat vaaleampia kuin viimeksi mainitun menetelmän antamat.