94. ROBERTS AND SMITH : SPECTROPHOTOMETRIC MEASUREMENTS [Vol. 86 Spectrophotometric Measurements of Theaf lavins and Thearubigins in Black Tea Liquors in Assessments of Quality in Teas* BY E. A. H. ROBERTS (Indian Tea Association, Butlw’s Wharf, London, S . E. 1) AND R. F. SMITH (The Laboratories, J . Lyons 6. Co. L.td., Kensington, London, W.14) The theaflavins and thearubigins formed as a result of enzymic oxidations of polyphenols during the fermentation process in tea manufacture determine the colour of a tea liquor and are associated with some of the other liquor characters recognised by tea tasters. A spectrophotometric method for determining theaflavin is described. This method also yields an approxima- tion of the total thearubigin content, and the ratio of optical-density values for thearubigins at 380 and 460 mp provides a further parameter giving an indication of whether lightly or deeply coloured thearubigins predominate.-4s a result of the fermentation process in tea manufacture the liquor of black tea develops certain characters not present in green or unfermented teas. Among the terms used by tasters to describe these characters are colour, strength, quality and briskness. It has been shown that these characters, measured organoleptically, are influenced by the extent to which fermentation has taken place, and, further, that colour and strength are correlated with the polyphenol oxidase activity and total polyphenol content of the plucked shoots from which the tea was manufactured.lS2 It follows that some, at least, of the liquor charac- ters of a black tea are due to the presence of polyphenolic enzymic oxidation products.The main polyphenolic oxidation products found in black tea extracts are the thear~bigins.~~~ These are a complex mixture of substances derived largely, if not entirely, from two parent substances, l-epigallocatechin and its gallic acid ester. These substances are not polymers, as previously thought, in fact they are probably mainly dimeric. They have fairly strongly acidic properties and in tea liquors considerable proportions of them are present as potassium and calcium salts. Although no pure substances have been isolated from the thearubigin mixture, some separation into fractions has been achieved. S I and S I1 fractions differ in their solubility relationships and in their chromatographic behaviour.3 The absorption spectrum of the S I1 fraction is similar to that of the S I fraction; it never- theless shows significantly greater absorption in the visible region, but not in the ultra- violet r e g i ~ n .~ The other oxidation products detected in black tea include theaflavin and theaflavin gallate, a bis-flavanol and its mono- and di-galloyl esters and several trace substance^.^ to 8 The theaflavin and theaflavin gallate have been isolated as pure substances and rather tentative structures have been suggested. The bis-flavanols show a single absorption band (Amax. = 280 m ~ ) ~ and contribute nothing towards the colour. Contributions by the trace substances may be disregarded, so that colour :in tea liquors may be considered as due to * Presented at the meeting of the Society on Tuesday, September 20th, 1960.February, 19611 OF THEAFLAVINS AND THEARUBIGINS IN BLACK TEA LIQUORS 95 theaflavins and thearubigins alone.Determination of these two groups of substances should therefore give a precise method of measuring the colour of a tea liquor, and such measurements might also be expected to be correlated with some of the other liquor characters. The method developed for determining theaflavins and thearubigins depends on the fact that the theaflavins are almost quantitatively extracted from a tea liquor by one extrac- tion with either ethyl acetate or isobutyl methyl ketone. These solvents do not extract thearubigins of the S I1 type, but there is a partial extraction of the free-acid forms of the S I type thearubigins.Potassium and calcium salts are not extracted. The thearubigins extracted by ethyl acetate or isobutyl methyl ketone are soluble in aqueous sodium hydrogen carbonate] whereas the theaflavins are insoluble. Complete separation of theaflavins and thearubigins is therefore effected by shaking the ethyl acetate or isobutyl methyl ketone extract with aqueous sodium hydrogen carbonate. Theaflavin and its gallate have well defined absorption maxima at 380 and 460mp.6*8 Either of these wavelengths is suitable for direct spectrophotometry in the extract washed with sodium hydrogen carbonate, as no other substances are present that absorb at these wavelengths. The fall in optical density, which results from the washing with sodium hydrogen carbonate, affords a method of determining the extractable thearubigins.Direct spectrophotometric determination of residual thearubigins in the aqueous layer after extraction with ethyl acetate or isobutyl methyl ketone is not possible, as a high pro- portion of the theambigin molecules is present as anions, which are more deeply coloured than the free acids. Addition of excess of aqueous oxalic acid reduces the colour intensity to that of the free acids, and spectrophotometry is possible after this acidification. METHOD PREPARATION OF THE TEA EXTRACT- In the traditional technique of tea tasting a weighed sample of tea is infused with boiling water for from 5 to 10 minutes in a porcelain pot fitted with a lid. The resulting liquor is decanted into a porcelain cup.This method was not sufficiently reproducible for analytical purposes and it was necessary to modify it; the modified method is described below. Weigh 9 g of tea into a 500-ml Erlenmeyer flask placed on a boiling-water bath. Bring 375 ml of distilled water to the boil, and pour it on to the tea. Allow extraction to continue for 10 minutes, without letting the temperature fall below 85°C. Filter through a plug of cotton-wool, and allow to cool just to room temperature. PROCEDURES FOR PARTITION AND SPECTROPHOTOMETRY- Shake 50 ml of the cooled, well shaken, filtered extract with 50 ml of isobutyl methyl ketone, taking care to avoid the formation of an emulsion. Allow the layers to separate, and dilute a 4-ml portion of the isobutyl methyl ketone layer to 25ml with methanol (solution A).Dilute a 2-ml portion of the aqueous layer to 10ml with water and then to 25ml with methanol (solution B). Shake 25 ml of the isobutyl methyl ketone layer vigorously for 30 seconds with 25 ml of a 2.5 per cent. aqueous solution of sodium hydrogen carbonate. Allow the layers to separate, and discard the aqueous layer. Dilute 4 ml of the washed isobutyl methyl ketone layer to 25ml with methanol (solution C). Add 2ml of a saturated aqueous solution of oxalic acid and 6ml of water to a 2-ml portion of the aqueous layer left from the first extraction with isobutyl methyl ketone, and dilute to 25 ml with methanol (solution D). Measure the optical densities] E,, E,, E, and ED, of solutions A, B, C and D, respectively, in 1-cm cells at 380 and 460 mp with a Unicam SP500 or similar spectrophotometer.NOTES-The two layers obtained from a 1 + 1 mixture of isobutyl methyl ketone and water are nearly equal in volume. With isobutyl methyl ketone there is no danger of the volumes of the layers being affected by partial saponification of the organic solvent, as might occur with ethyl acetate, particularly if recovered solvents are used. In the preparation of solutions B and D from the aqueous phase from the first partition, it is essential that the methanol content should not exceed 60 per cent. otherwise a cloudiness may develop, owing to precipitation of pectins. If the sodium hydrogen carbonate contains more than a small amount of sodium carbonate, theaflavins may be Iost by alkaline autoxidation during washing of the isobutyl methyl ketone extract.Analytical-reagent grade sodium hydrogen carbonate should therefore be used, and the solution should be freshly prepared. To minimise the possibility of losses by autoxidation the period of shaking96 ROBERTS AND SMITH SPECTROPHOTOMETRIC MEASUREMENTS [Vol. 86 must be as short as possible, and one layer should be completely removed from the other immediately after they have separated. Under normal conditions solution C is stable, but the measurement of its optical density should not be delayed. How- ever, the &/ED ratio has a particular significance, which will be referred to in subsequent publications. EVALUATION OF THEAFLAVIN CONTENT- There is always considerably more theaflavin gallate than theaflavin in a tea, so that little error will be introduced by expressing total theaflavins as theaflavin gallate.The optical densities, E,, at 380 and 460 mp are converted into a percentage of anhydrous theaflavin gallate by multiplying by the factors 2-25 and 6.69, respectively. These factors are applicable only when the conditions of extraction, partition and spectrophotometry are as described above, and are calculated from the optical-density values for theaflavin gallate shown in Table I. It is assumed that the theaflavin gallate used for standardisation purposes is the dihydrate of molecular weight 892-7.s In routine determinations of theaflavin and thearubigins no use is made of the E, values. NOTE-The ratio of optical densities at 380 and 460mp should be 2.98 to 1.If the ratio is appreciably greater than this, incomplete removal of thearubigins by sodium hydrogen carbonate is indicated. A lower value would suggest that the ratio of theaflavin to theaflavin gallate was greater than usual (see Table I). EVALUATION OF THEARUBIGIN CONTENT- Any factor for converting optical density into a percentage of thearubigin must be somewhat arbitrary, as we are dealing with a variable and rather complex mixture of sub- stances, none of which has been isolated in a pure state. A further complication is introduced by the uncertainty as to the degree of hydration of the thearubigins. It is apparent from Table I that the optical densities for the S I and S I1 fractions differ appreciably at 460 mp, but do not differ much from each other at 380mp.The average value of EY*:Zh at 380 mp for the two fractions is 0.733. Optical densities at 380 mp obtained for these two fractions with four other teas were- Sample No. .. .. .. 1 2 3 4 Optical density of S I fraction . . 0.712 0.655 0.658 0.739 Optical density of S I1 fraction . . 0.734 0.796 0.720 0.810 The acceptance of 0-733 as an average value of EP':Eh at 380mp for total thearubigins is therefore unlikely to lead to any considerable error, and, if this average value is assumed, the percentage of extractable thearubigins in a tea is, for the conditions of extraction, partition and spectrophotometry described above, approximately 7.06 (2E, + E, - Ec). The results must be accepted with certain reservations, but they are of the expected order of magnitude.THE ESIO/EIIO RATIO FOR THEARUBIGINS- The ratio of the values for 2E, + E, - E, at 380 and 460 mp in different teas has varied from 3.6 to 8.2. This indicates that the mixture of thearubigins in teas is by no means constant. If it is assumed that thearubigins have approximately the same optical densities at 380 mp, the variation in this ratio gives some indication of the average intensity of colour (at 460mp) of the thearubigins. A high ratio implies relatively light colour, and a low ratio a correspondingly deeper colour. As will be apparent from Table I, the more deeply coloured S I1 fraction has a lower ratio than the corresponding S I fraction. TABLE I E",Eh VALUES FOR THEAFLAVIN, THEAFLAVIN GALLATE AND THEARUBIGIN FRACTIONS The absorption spectra of the theaflavins and the thearubigin fractions were plotted over the range 220 to 600 mp.The resulting absorption curves and optical densities at certain wavelengths have been published previously5 ~8 E P ' ~ ~ ~ value at- Sample r - - - A - , Ratio 380 mp 460 mP E380/E460 Theaflavin . . .. .. . . 3400 1.235 2.75 Theaflavin gallate . . . . . . 2.225 0.747 2.98 Thearubigins (fraction S I) . . 0-717 0-138 5.20 Thearubigins (fraction S 11) . . 0.750 0.233 3.20February, 19611 OF THEAFLAVINS AND THEARUBIGINS IN BLACK TEA LIQUORS 97 This ratio is a useful extra parameter in describing the thearubigins of a tea, for thearubigin content is not necessarily proportional to depth of colour, particularly if the E380/E@) ratio is high. A rough estimate of the percentage of thearubigin in such teas is given by 4 x 7-06 (2E, + E, - Ea) when optical densities are measured at 460 mp.In the earlier stages of this investigation measurements were carried out at 460mp only, but, so long as the teas originated from Assam, the optical densities could be used to give an approximation of their thearubigin contents. With nearly all Assam teas this ratio falls between 3.6 and 4.4 (average 4.0). RESULTS Many commercial and experimentally manufactured teas have been analysed by the proposed procedure and detailed results will be reported elsewhere. Typical results are considered below. Those in Table I1 give some idea of the range of values obtained. TABLE I1 REPRESENTATIVE ANALYSES OF COMMERCIAL TEAS Thearubigin contents in brackets were calculated from optical densities at 460 mp Theaflavin Thearubigin Sample content, content, Ratio Taster’s remarks % % E,*O/E4liO N.E.India- - Golden colour, strong - Bright - Coloury, dull, common Good quality (C) . . .. 0.78 (8.9) Medium quality (C) .. 0.68 (7.6) Poor quality (C) .. 0.36 (7.1) Duars O.F. grade (C) . . 0-58 7.6 4.38 - 4-24 - Assam O.F. grade (Cj . . 0-70 13.1 Assam B.P. grade (CTC) . . 1.45 16.7 3.64 Very bright, golden Duars (Legg cut) . . .. 1.08 14.7 4.57 Hard, bright colour, with P.F. grade (C) . . .. 0-92 14.6 8.25 Bright, golden B.O.P.F. grade (C) . . . . 0.81 17.1 7.33 Rich, very coloury B.O.P. grade (C) . . .. 0.42 13-6 6.00 Dull B.P. grade (C) . . .. 0.34 8-5 - Thin, grey P.F. grade (C) . . . . 0.23 15-0 4.53 Very thick, dull, muddy rich colour Ceylon- Nyasaland- Kenya- Argentine- C = Conventional manufacture.CTC = C.T.C. manufacture. The thearubigin content of a tea is always considerably higher than the theaflavin content, but, as theaflavins are much more intensely coloured (see Table I), their contribution to total colour is a decidedly significant one. In assessing tone of colour, the taster is influenced more by the theaflavin content than the thearubigin content, and in conventionally manufactured teas the preferred liquor colours are associated with theaflavin contents of 0.75 per cent. or more. Conventional methods of manufacture, particularly in N.E. India, have to some extent been replaced by other methods (C.T.C. and Legg cut) in which the leaf receives a more thorough bruising, resulting in a quicker and more extensive fermentation.Analyses of such teas show increased theaflavin and thearubigin contents and an increase in the ratio of theaflavin to thearubigin. This accounts for the increased “brightness” and greater depth of colour normally associated with these methods of manufacture. Table I1 also shows the different E380/&60 ratios obtained for thearubigins. These are lowest in teas originating from N.E. India. Theaflavin and thearubigin contents are also affected by fermentation conditions, as indicated in Table 111. It will be noted that the theaflavin content reaches a maximum level comparatively early in the fermentation process, and that, in the later stages of fer- mentation, thearubigin contents increase slowly a t the expense of the theaflavins.As the liquor characters of strength, briskness and quality are affected by variations in the duration of fennentation,l it is considered probable that these particular characters are deterrnined, to some extent at least, by the theaflavin and thearubigin contents.98 SHANKARANARAYANA AND PATEL : THE VOLUMETRIC v o l . 86 TABLE I11 EFFECT OF DURATION OF FERMENTATION ON THEAFLAVIN AND THEARUBIGIN CONTENTS OF C.T.C. MANUFACTURED TEAS Each result is an average from six separate experimental manufactures Fermentation time, hours . . .. 1 2 3 4 5 Theaflavin content, % . . . . 1.61 1.46 1.34 1.27 1-17 Thearubigin content, % . . . . 13.0 16.2 16.6 16.7 17.1 CONCLUSIONS We are of the opinion that the measurement of theaflavins and thearubigins, coupled with the E,,,,/E,,, ratio for thearubigins, represents the best available method for determining colour in tea liquors.In view of the probable association of these variables with other liquor characters, it might be thought that these methods would serve as a basis for a chemical evaluation of the market value of a tea. Quality in tea is not determined by polyphenolic oxidation products alone, for there are other im- portant factors, among which caffeine and the volatile substances responsible for aroma may be mentioned. Market valuations are also affected by such factors as appearance and keeping properties on storage. The replacement of traditional methods of tea tasting by chemical analysis is not yet therefore in sight, but it is claimed that the methods described above should prove extremely useful in supplementing a taster’s report. Analytical results, unlike a taster’s evaluation, are not affected by market fluctuations, and are much more suitable when it is desirable to maintain records of the properties of teas manufactured. The analytical method may also be expected to be developed as a means of control during manufacture. We thank the Indian Tea Association (London) and Messrs. J. Lyons & Co. Ltd. for permission to publish this paper. This, however, is too optimistic a view. REFERENCES 1. 2. 3. 4. 5. 6. 7. -,- , Ibid., 1959, 10, 172. Roberts, E. A. H., J. Sci. Food Agric., 1958, 9, 381. Roberts, E. A. H., in preparation. Roberts, E. A. H., Cartwright, R. A., and Oldschool, M., J. Sci. Food Agric., 1957, 8, 72. Roberts, E. A. H., Ibid., 1958, 9, 212. Roberts, E. A. H., and Williams, D. M., Ibid., 1958, 9, 217. Roberts, E. A. H., and Myers, M., Ibid., 1959, 10, 167. 0. -, -, Ibid., 1959, lo, 176. Received October SEith, 1960