V O L U M E 23, NO. 2, F E B R U A R Y 1 9 5 1
J 100
I
'
327
4 - 9
u
YI f
0
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I
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E
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7
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TR70, "C. Figure 11. Hardness after Storage at -45' TR7O
TR70, " C . C. vs.
Figure 12.
0 Butadiene-isoprene-styrene
0 Butadiene-isoprene-styrene co- and terpolymers
00-
Hevea (50 parts channel black) 0 Hevea (gum)
Hevea (50 parts channel black) 0 Hevea (gum)
Most of the stocks were based on butadiene-styrene copolymers polymerized a t 41' and 122" F., respectively, and contained various proportions of styrene. Tread stocks based on Hevea, butadiene-isoprene copolymers, butadiene-isoprene-styrene terpolymer, and a gasket stock containing no black based on Hevea were included. Durometers were measured a t intervals over 12-day periods a t -35" and -45' C., respectively. Hardness of all the stocks with the esception of the Hevea stocks correlated fairly well with TR70 at both -35" and -45' C. Hardness of the Hevea stocks did not correlate with TR70 a t either temperature. The smoked sheet tread compound attained approximately equivalent hardness a t -35" and -45' C. The smoked sheet gasket compound was about as hard as the tread compound when stored for 12 days a t -35" C., but when stored for a similar period a t -45" C. the gasket compound hardened very little whereas the tread compound became even harder than a t -35' C. The difference between the correlation of the Hevea compound n i t h TR70 a t -35' C. in the hardness test and the compression set test discussed previously can be explained by assuming less crystallization in the hardness test due t o the absence of strain \vhich increases tendency to crystallize.
Hardness after Storage at -35' TR70
C.
us.
and terpolymers
ACKKOW LEDGMENT
The authors wish l o express their thanks to P. R. Van Buskirk and A. S. Weisser for their assistance. This work was carried out under the sponsorship of the Chcmicals and Plastics Section, Research and Development Branch, Office of the Quartermaster General. LITER4TURE CITED
(1) Am. Soc. Testing Materials, A.S.T.51. Designation D 599401'. (2) Beu, K. E . , Reynolds, W.B., Fryling, C. F., and Slurry, H. L., J . Polymer Sci., 3, 465 (1948). (3) Gehman, S. D., Woodford, D. E., and Wilkinson, C. S., Jr., Ind. Eng. Chem., 39,1108 (1947). (4) Gibbons, W. A., Gerke, R. H., and Tingey, H. C., IND. ENQ. CHEX.,ANAL.E D . ,5 , 2 7 9 (1933). (5) Hart, E. J., and Meyer, A. JT.,J . Am. Chem. Sac., 71, 1980 (1949).
(6) Lucas, V. E., Johnson, P. H., Wakefield, L. B., and Johnson, B. L., Ind. Eng. Chem., 41, 1629 (1949). (7) Svetlik, J. F., private communication through A.S.T.M. (8) Yereley, F. L., and Fraser, D. F., Ind. Eng. Chem., 34, 332 (1942). RECEIVEDOctober 16, 19.50. Presented before the Division of Rubber C H E M I C ~SOCIETY, L International Meeting, CleveChemistry, AWERICAV land, Ohio, Oatober 11 to 13, 1950. Contribution 109 from General Laboratories, United States Rubber Co.
Determination of Trigonelline in Goff ee R . G. MOORES AND DOROTHY >I. GRENINGER, General Foods Corp., Hoboken, S. J .
Trigonelline represents about 5% of the soluble solids in coffee beverage. It contributes to coffee flavor and aroma, and may have important physiological properties. A n easy and reliable method for determining trigonelline was needed in the study of coffee composition and flavor development. In the new method, trigonelline is purified by chromatography on a solid adsorbent column, and is measured by ultraviolet absorption spectrophotometry. This procedure is more reliable and convenient than earlier methods. The method will be useful in further studies on the composition and processing of coffee, as well as in physiological studies involving closely related compounds such as nicotinic acid.
T
HE presence of trigonelline in green coffee was first reported by Polstorff in 1909 ( 2 1 ) , and later verified by Gorter ( 6 ) . I t is the methylbetaine of nicotinic acid with the ~ t r u c t u r a l formula: H I
I
CH, Trigonelline represents about 5% of the water-soluble portion of roasted coffee. It has a bitter taste about one fourth that of caffeine. Trigonelline may be considered the source of pyridine found in coffee aroma. Hughes and Smith (9) found pyridine
ANALYTICAL CHEMISTRY
328 and nicotinic acid among the decomposition products of trigonelline when it was heated in a sealed tube a t 150" to 220" C. The structure of trigonelline suggests the possibility of its demethylation in vivo to nicotinic acid, the antipellagra factor. However, the physiological properties reported in the literature are conflicting and lead to no certain conclusions regarding its tlietaryrole (3, 7 , l 4 , 1 8 ) . A reliable analytical procedure for trigonelline is important becauw of its contribution t o coffce flavor and aroma and its possible biological Pignifirancc. WRES FOR TRIGONELLINE
Several niethodt for t,lie drtermination of t.rigonellinc have been described in the literature (4,12, 19, 22, 23). Their deficienries are discussed briefly in the following paragraphs. Precipitation Methods. Thc trigonelline method of Nottbohm and Mayer (19) involves precipitation of trigonellinc n-ith iodine from a chlorogenic acid and caffeine-free coffec extract, and titration of the iodine coniples in alcohol with thiosulfate. The iodine precipitation requires a high concentration of trigonelline for quantitat,ive results. The method can be applied only t o green coffec, because the iodine salt does riot crystallize itrtdily in the presence of roasted coffee hnstituents. Methods have been reported for the determin:ition of I)isinuth based on the precipitation of alkaloid iodobisniuthates (6, 16). Trigonelline was found to precipitate quantit,atively, 011 an equal molar basis, with t,his reugc,nt. This method gave good results hut satisfact,ory precipitation could not be obon green trtined on coffee. h mod 1 of the Rlott,:i and Seisser proctdure ( 2 a ) was used previouP1y for trigoiielline analysis in thip laboratory. This method, which in~olvesa phosphotungstic acid precipitation of the trigonelline from :i lead-clarified water ext,ract and alkaline oxidation of thr regenerated trigonelline, is inconvciiicnt and is subject t o error.. that, h i i t its reliability. Colorimetric Methods. The measurcnient of trigonelline would be simplified by ronversion to a soluble colored product, but none of the conversion techniques tried in this n.ork ivew quantitative. Colorimetric nictliods suggested in the literature for the estimation of pyridine ring compounds depend upon the formation of Schiff's bases from the corresponding glutjaconaldehydes (12, 13). However, this reaction cannot he applied dircctly t o the pentavalent nitrogen in trigonelline. Saret,t, Perlzweig, and I,evy (BO, 22) found that :ilkdine hydrolysis of t,rigonelline in the presence of a source of animonia yielded a substance that develops a color with cyanogen bromide and amines; t,he trigonelline presumably had been converted to nicotinic acid, However, they found that this conversion amounted t,o only 70%. 11 lit work by Huff (8) IIRR shown t'hat the conversion of trigonelline to nicotinic acid is not quantitative: under the most iavora1)le conditions t,lir nicotinic acid isolated was only SOY0 of the theoretical yield. Kodicek and \Tang ( 1 2 )and Fox, lIcSeil, and Field (4)measured trigonelline hg the color of the compound formed when benzidine or dianisidine is added to the g1utacon:ildehyde formed Preliminary analyses iri this 1:iborator)- on pure trigonelline solut,ions by a slight modification of t,he dianisidine method were unsatisfactory. Th(, timc for developnieiit of musimuni color varied from 14 t o 37 minutes. Iteproducibility of maximum readings was poor; when 4 mg. of t,rigonelline w r e hydrolyzed, maximum absorbancy i i i e a ~ ~ r c m ~ varied i i t s from 0.724 to 1.072. T h e addit,ion of urea as a buffer in the neutra1izat)ion did not improve reproducibility. With a p H range of 6.35 to 8.47 a t the time of the addition of the color reagents, the highest absorbancy reading was obtained a t p H 8.2. After 24 hours' standing, solutions of hydrolyzed trigonelline gave less color mith the reagent, and absorbancy readings a-cre not proportional to concentration.
Spectrophotometric Properties of Trigonelline. Following the development of satisfactory spectrophotometric methods for determining chlorogenic acid ( 1 7 ) and caffeine (11) in coffee, absorption measurements were applied to trigonelline. Trigonelline in water solution has a maximum absorption a t 264.5 and a niininiuni at 240 nip (Figure 1). Absorbancy of aqueous trigonelline solutions is proportional to concentration and is constant over a p H range of 4 to 8. Other substances in coffee have absorption in this region-e.g., caffeine, chlorogenic acid, proteins, etc. S o chemical reagent could be found t,h:tt, selectively separated trigonelline without itself absorbing light in the same region. The unsatisfactory result,s obtained in the use of known color and precipitation reagents indicated t'he need for an entirely different approach for the purificat,ion and estimation of trigonelline. Several adsorbents, such as Zeokarb H, 3 - L clay, Lloyd's reagent, zeolite, Amberlite IIt-100, and Filtrol, remove trigonelline from pure solution. The first three adsorbents contain soluble materials which absorb light a t 265 nip and cannot be removed by any reasonable amount of washing. Both t,hc percolat,e and the ammonia eluat,e from zeolite contain nontrigonelline material which absorbs light at 265 nip. . h b e r l i t e IR-100 can be washed fairly free of the int,erferingmaterials, but, elution of the trigonelline c:iiinot I w made quantihtive. Wibh Filtrol, adsorption of the t,rigonelline from a n alcohol-water solution and elut,ion with ammonium hydroside are quant,itat,ive(Table I). These solvents do not remove any material that interferes with spectrophotometric, measurements. Standard Trigonelline Samples. Two samples of trigonelline were used in this work. One sainple was prepared by the methvlation of nicotinic acid, using the method of Winterstein and Weinhagen ( 2 5 ) . I t was identical in ultraviolet absorption and iodine equivalent 1vit.h a reference standard obtained from the S.1I.A. Corp., Chagrin Falls, Ohio. The reference st,andard had a nitrogen content of 10.27% by the Dumas method. The theoretical value is lO.22a/,. -it 264.5 mp, the wavq length of
WAVELENGl w
IMILLIMIGRONS)
Figure 1. Absorption Curves for Trigonelline before and after Filtrol -4bsorption 1. Pure trigonelline 2. Trigonelline after adsorption and elution from Filtrol 3. Blank
V O L U M E 2 3 , NO. 2, F E B R U A R Y 1 9 5 1 T a b l e I.
Recovery of Trigonelline b y Adsorption a n d E l u t i o n from F i l t r o l
Sample Description Pure trigonelline
Mixture of trigonelline, caffeine, and chlorogenic acid
Trigonelline Recovery in Eluate by Ultraviolet .4bsorption, % 104.0 102.1 100.2 104.2 101.7 105.4 101.2 103.2 101.9 99.7 98.4 99.5
97.3
Mixture of trigonelline, caffeine, chlorogenic acid, and nicotinic acid Pure trigonelline added t o green coffee extract
100.0 98. o a 101.2'L 100,s 100.0
Pure trigonelline added to roasted coffee extract
98.9 98.9 a Corrected for nicotinic acid on basis that 100% of total nicotinic acid was in eluate.
maximum absorptioii, both samples of trigonelline had an of 297. ULTRAVIOLET ABSORYTIOY METHOD FOR TRIGONELLINE
Reagents and Equipment. Ethyl alcohol, 95 and 50% by volumc. Sulfuric acid, 2y0 by volume in 9570 ethyl alcohol. Ammonium hydroxide, 3% by volume, 30 ml. of 28.7y0 animonium hydroxide per liter. Potassium permanganate, 1% by weight in water. Sodium sulfite, 5% by weight in water. Potassium ferrocyanid?, 106 grams of KrFe(CS)a.:3H20dissolved in water and diluted to 1 liter. Zinc acetate, 219 grams of Zn(C2H302)2.2Hz0 and 30 ml. of glacial acetic acid dissolved in Lmter and diluted to 1 liter. - ilcetic acid, C.P.glacial. Filter paper, Green's fluted S o . 488l/2; 15-cm., Khatman S o . 3.
Celite, Yo. 545, and analytical (Johns-Manville Corp., S e w York, K. Y.). Filtrol, Super Filtrol (Filtrol Corp., Los Sngeles, Calif.). Prepare adsorption mixture by thoroughly mixing 2 parts of Celite S o . 545 and 1 part of Filtrol. Extraction and adsorption tubea. ,It.tach 80-mm. lengths of 6-mm. glass tubing to the bottom of 18 X 150 nim. test tubes. Beckman quartz spectrophotomet'er, Model DU. Sample Preparation. Coff ec samples are prepared for :tiiiilysir by grinding and flaking as for chlorogenic acid ( 1 7 ) . Extraction of Green and Roasted Coffee. Weigh accurately 2 grams of sample and mix with 4 grams of Celite S o . 545. Transfer to an extraction tube containing a glass wool plug and Celite No. 545 filter bcd. Place the extraction tube in a 500-ml. filter flask connected to a vacuum line, Percolate 200 ml. of 507, ethyl alcohol through the sample a t a uniform rate of about 7 nil. per minute, transfer thc extract to a 200-ml. volumetric flask, and make to volume. Adsorption and Elution of Trigonelline. Prepare a Filtrol adsorption column by placing a glass wool plug in the bottom of the tube and covering with a Cclite So. 545 bed about 10 mm. thick. On top of the Celite bed, place about 6 grams of the 2 to 1 Celite-Filtrol mixture. Tap the tube, connect to a 500ml. filter flask, and apply vacuum to compress the column. Percolate through the tube 75 ml. of 2 N sulfuric acid in about 10 minutes. Transfer 100 ml. of the coffee extract to a 250-ml. separat,ory funnel equipped with a rubber stopper or ground-glass joint to fit the top of the adsorption tube. After the acid wash of the column is complete, connect to the adsorption tube the separatory funnel holding the coffee extract. Draw the coffee extract through the Filtrol column a t a uniform rate of about 5 ml. per minute. As soon as the extract is drawn through, wash the column successively with 50 ml. of 5OYO ethyl alcohol, 50 ml. of 2 % sulfuric acid in 95% ethyl alcohol, and 50 ml. of 95y0 ethyl alcohol. Some liquid should be retained on top of the adsorption column a t all times to avoid channeling.
329 Remove the filter flask containing the trigonelline-free extract and wash solutions, and replace it with a clean 250-ml. filter flask. Elute the adsorbed trigonelline by percolating through the column 150 ml. of ammonium hydroxide. Transfer the eluate to a 200-ml. volumetric flask and dilute to volume w-ith water. The time for the elution should be about 20 minutes, making the total time for adsorption, washing, and elution about 90 minutes. Measurement of Trigonelline in Green Coffee. Transfer 50 ml. of the eluate to a 100-ml. volumetric flask and dilute to volume. Measure the absorbancy (absorbancy or A , = optical density) of the diluted eluate a t 264.5 and 325 mp. Subtract the absorbancy at 325 from the absorbancy at 264.5 nip and from this difference calculate the trigonelline content, using an value of 297. Measurement of Trigonelline in Roasted Coffee. Place 50 nil. of the eluate in a 100-ml. volumetric flask, add 10 ml. of potassium permanganate solution, mix, arid let stand 10 minutcs. Add 3 ml. of sodium sulfite solution with stirring, then 1.5 ml. of acetic acid, and finally titrate with sulfite aolution to the disappearance of the niangancse dioxide precipitate. RI ake to volume and filter through No. 5 Whitman paper, discarding the first portion. Measure the absorbancy a t 264.5 mp. Place a separate 50-ml. aliquot of the eluate in a 100-ml. volumetric flask, and add about 25 nil. of water and 1 ml. of acetic acid. Add 5 ml. of zinc acetate solution and mix; then with swirling add 5 ml. of potassium ferrocyanide solution. Make to volume and shake thoroughly. After 3 to 5 minutes filter through a dry folded paper, such as Green's N o . 4881/2. Discard the first 5 to 10 ml.and collect enough to fill an absorption cell. Measure the absorbancy a t 264.5 mr. Subtract this from the previous absorbancy reading and calculate the trigonelline content using this corrected figure. DISCUSSION
Sample Preparation of Trigonelline Extraction. The iinport a m e of proper sample preparation has been demonstrated in t,he current work. Ersentially complete cell rupture, as provided by flaking, is necessary for reliable analysis of plant material such as coffee. 1,ktraction of trigonelline with SO?;', ethyl alcohol is more satisfactory than extraction with 95% ethyl alcohol or water. 1i:xtractiori with 9570 ethyl alcohol is extremely slow, \vhile watcr extracts percolate very .ilorvly through Filtrol columns. Adsorption and Elution with Filtrol. Linder proper conditions of percolation, washing, and elution, the Filtrol column can be used to separate trigonelline quantitatively from caffeine and chlorogenic acid in pure solutions (Table I). I n the concentrations normally present in coffee extracts, caffeine is adsorbed on the column but not eluted by the ammonia. Chlorogenic acid is adsorbed on Filtrol and is eluted with ammonia. However, when the adsorbcnt is washed with acid before percolat,ion of the alcohol extract, the adsorption of chlorogenic acid is reduced, and washing with acid-ethyl alcohol after the percolation effectively elutes the small amount, of chlorogenic acid adhering to the column. Washing with acid-ethyl alcohol does not remove the trigonelline, hut does remove some of the ammoniaelutable substances present in roasted coffee extracts which absorb light at and below 325 mp. Hughes and Smith (9) reported that only 1 to 3y0 of the trigonelline loss on roasting could be attributed to nicotinic acid formation. The present work substantiates their data by finding little or no nicotinic acid when trigonelline was roasted alone or in mixtures of coffee constituents. These observations are based on results of alkaline iodine titrations. Sicotinic acid does not react with iodine under the conditions used for trigonelline analysis. Sicotinic acid is adsorbed and eluted along with trigonelline in the proposed met'hod and has an absorption maximum near 265 mp. Trigonelline in Green Coffee. The trigonelline content of green coffee by the spectrophotometric method is about 1OY0 lower than that obtained by the original Slotta-Keisser procedure (Table 11). The absorption figures have been substantiated by applying the Slotta-Seisser iodine titration to the ammonia-free eluates from the Filtrol column. Good agreement between the
330
ANALYTICAL CHEMISTRY
absorption measurements and the iodine titrations indicates better selectivity in the present isolation technique. The correction for the absorption reading.at 325 mp is used as the alternative to a blank sample correction (Figure 2). This measured absorption is apparently caused by light-scattering substances washed from the Filtrol column. I n the present method the effect of these substances on the absorbancy measurements is sufficiently constant between 265 and 325 mp to make feasible use of the latter reading as a correction for the sample absorbancy a t 264.5 mp. This correction amounts to no more than 0.05 absorbancy unit. Absorption measurements on the eluates before and after treatment with zinc ferrocyanide substantiate further the specificity of the present method for trigonelline in green coffee. This reagent precipitates trigonelline quantitatively from water solutions without interfering with the light absorption a t 264.5 mp. The difference between the total absorption and the absorption removed from the eluates by zinc ferrocyanide treatment is equal to the blank (Figure 2), so that the trigonelline content is essentially the same when calculated using a blank correction or on the basis of absorption removed by ferrocyanide treatment (Table
.700
-
,600-
SO0
-
0 >
5
.400-
m 0
m 4
.300
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Table 11.
SlottaNeisser Method,
% Santos
Spectrophotometric Method Total Aa of Total As A Sremoved eluate at minus by 264.5 mp, blanks, precipitationc,
%
%
%
1.32
1.33
Av. Guatemalan
Av. Colombian
1.33 1.11 1.11
1.11
1.06 1.06
Av.
WAVELENGTH
Trigonelline Content of Green Coffeea
1.06
1.29
1.22
.02 .03
0.98 0.99 0.98 0.98 1.05 1.05 1.01
,o:!
1.22
...
... ... ...
.02 .09 .09 1.05 1.00 1.01 0.98 0.99 1.14 1.15
0.95 0.96 0.94 0.94 1.03 1.03
1.03 1.03
1.05
0.96
1.03
1 .OO 0.99 1.00
...
...
...
...
All results expressed on dry weight basis. b Trigonelline calculated from total absorbancy, A,, of eluate at 264.5 mp minus absorbancy at 325 r n p . C Trigonelline calculated from difference between total absorbancy of eluate and that remaining after zinc ferrocyanide treatment. a
Trigonelline in Roasted Coffee. Roasting plant materials generally increases their chemical complexity and magnifies analytical difficulties. Coffee is no exception, and extensive experimental work was required before the spectrophotometric method could be applied to roasted coffee. Experiments were designed to study the effect of roasting on trigonelline alone and in the presence of other coffee constituents, in order to define the nontrigonelline materials which absorb light a t 264.5 mp. Heating pure trigonelline with 12% added water in a sealed glass tube a t 180" to 187" C. for 63 minutes causes a significant change in its light absorption behavior. The alcohol extract of the roasted trigonelline ha8 no peak near 265 mp, but has fairly high absorption in the entire spectral region from 240 to 325 mp. Iodometric titration directly on the alcohol extract after evaporation shows an apparent trigonelline loss of 83%. This alcohol extract of the roasted trigonelline contains no trigonelline by the ultraviolet absorption method. Because analyses on roasted coffee do not show such a drastic change in the nature of the tri-
Figure 2.
(MILLIMICRONS)
Absorption Curves for Trigonelline in Green Coffee Extracts
1. After adsorption and elution on Filtrol, no treatment 2. Absorption removed from eluate by permanganateferrocyanide treatment 3. Eluate after permanganate-ferrocyanide treatment
gonelline, the presence of other materials in coffee must prevent this complete degradation of trigonelline during roasting. A mixture containing chlorogenic acid, sucrose, green coffee dialyzate, caffeine, and potassium phosphate buffer a t p H 5.85 was roasted with and without added trigonelline. Direct spectral absorbancy readings at 264.5 mG on alcohol eytracts of the roasted products indicated only a 10% loss of trigonelline on roasting. When the alcohol extracts were carried through the adsorption-elution with Filtrol, spectrophotometric measurements indicated a loss of 68% of the trigonelline. Iodometric titration of these Filtrol eluates showed a trigonelline loss of 69%, which substantiates the ultraviolet measurement. Because nicotinic acid does not react with iodine under these conditions, the agreement between the ultraviolet and iodometric measurements indicates that little if any trigonelline has been converted to nicotinic acid during the roasting. The absorbancy a t 264.5 mp in the Filtrol eluates from roasted simulated coffee extracts containing no trigonelline was negligible, indicating no interference from the other constituents. High transmittancy in all the eluates from 285 to 325 mp indicated that no light-scattering materials were interfering. It is apparent that when trigonelline is roasted pure, or in a mivture of soluble green coffee constituents, substances are formed which absorb light a t 264.5 mp, but these substances are largely separated from the trigonelline in the adsorption-elution. Direct ultraviolet absorption on the ammonia eluates from Filtrol columns (Table 111)showed an apparent increase in trigonelline during the roasting of coffee. This apparent increase and the shape of the absorption curve indicated that the absorbancy of the roasted coffee eluates was not due entirely to trigonelline and that further purification was necessary. Because not all of these interfering substances absorb at 325 mp, this absorbancy cannot be used as a correction for roasted samples. Potassium permanganate has been used as a purification reagent in methods for determining caffeine in coffee (1, &, If, 16). This reagent in alkaline, neutral, or acid solution was found to have no effect on the ultraviolet absorption of pure trigonelline.
V O L U M E 23, NO. 2, F E B R U A R Y 1 9 5 1
331
Permanganate-sulfite treatment of roasted coffee eluates decreases appreciably the 265 mp absorbancy and largely eliminates the discrepancy between the absorbancy curves of the eluate and pure trigonelline solution. A zinc ferrocyanide precipitation of trigonelline has been used to determine the specificity of the absorbancy of the permanganate-sulfite treated eluates. When zinc acetate-potassium ferrocyanide treatment is applied to roasted coffee eluates, there remains some absorbancy in the region 265 to 325 mp (Figure 3). The residual absorbancy is the same when this clarification is applied directly to the eluate as to the permanganate-sulfite treated solution. It is more convenient to use the eluates before permanganate-sulfite treatment. Although the interfering material has not been identified, it is not trigonelline, and the residual absorbancy has been subtracted as a blank in the trigonelline determination for roasted coffee. The trigonelline analysis in roasted coffee depends upon the specificity of the Filtrol adsorption and elution, the removal of major interfering materials by mild oxidation, and selective precipitation of the trigonelline.
Table 111. Trigonelline Content of Roasted Coffee” Roasting w t . Loss Excluding Hs0, To
Green Coffee 9.2
Santos
.4v. Guatemalan
9.8
“4
10.2
AT.
.‘
0.89 0.89
1.70 1.53
1.15 1.03
0.89
1.61
1.09
0.76
1.84 1.84
1.16 1.16
0.75 0.72 0.72
1.84
1.16
1.74 1.78
1.12 1.16
0.72
1.76
1.14
0.74
Av.
Colombian
Slotta-
Seisser Method,
Spectrophotometric Method Aa Total Total removed 9 aat A8 by 264 5 minus precipimp, blankb, tationc, OI % /O % 1.13 1 16 1.17 1.19 1.16 0 97 0.94 0.94 0.95
0 92 0.92 0.92 0.92
All results expressed o n dry weight basis. b Trigonelline calculated from total absorbancy of eluate a t 264.5 m p minus absorbancy at 325 rnp. C Trigonelline calculated from difference between total absorbancy of eluate and t h a t remaining after zinc ferroryanide treatment. a
ACKNOWLEDGAIENT .7 0 0
-
.600
-
These methods for measuring trigonelline were developed a8 part of the program of fundamental research on the chemistry of coffee sponsored by the I\laxwell House and Sanka Divisions of General Foods Corp. The authors acknowledge with gratitude the interest and suggestions contributed by H. M. Barnes and I. I. Rusoff of this laboratory.
.500-
LITERATURE CITED >
9
.400-
m 0 K
m
.aoo-
.200-
_I0 0
-
0
220
I
240
I
260
I
260
W4VELENGTH
Figure 3. 1. 2.
3.
I
300
I
320
340
IMILLIMICRONS)
.4bsorption Curves for Trigonelline in Roasted Coffee Extracts
Eluate after permanganate treatment Absorption removed from permanganate treated eluate by ferrocyanide treatment Eluate after permanganate-ferrocyanide treatment
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Hughes and Smith (9, 10) have reported that trigonelline is lost during roasting and that the amount lost varies with the time and temperature of roasting. I n the present work the average decrease in trigonelline for three samples of coffee with an organic roasting weight loss of 9.7% was 39% by the Slotta-Xeisser method and only 15% by the spectrophotometric method (Table 111). This difference can be explained by the failure of the phosphotungstic acid to precipitate trigonelline completely from roasted coffee extracts. Apparently some of the products formed during coffee roasting interfere with this precipitation and also with precipitation by iodine or iodobismuthate. The spectrophotometric method, in which this interference is not a factor, gives a higher and more accurate measurement of trigonelline in roasted coffee.
RECEIVED July 26, 1950.
