1118
ANALYTICAL CHEMISTRY Table I.
Results of Experimental Data '3 Elemental
Analysis % Labile S Calcd. Found Calcd. Found
Dei-lation
C H
73.07 73.30 5.62 5.39 21.31 2 1 . 1 9
14.21 14.28 14.30
4-0.07 i - 0 09
?I
74 64 7 4 . 8 3 6.71 6.83 18 65 18.75
12.43
1% 35 12 32
- 0 08 -0.11
63.35 6 5 . 3 0 5.88 5.78 16.33 16.44
10 89
10.87 10 91
- 0 02 -0 02
Triazene 1.3-Diphenyltriazene CirHiiSs
K
1,3-Di(p-tolyl)triazene
CIIHI&;I
€1 S
1,3:Di(4-methoxvphen~l~tiiaaene CilHi602X3
C H
S
wo Pur1ty Above procedure
Polaiographicallj-
Ui.6
3i.6
Colilnwrcial triazene R
R
C->.-S=S if
=
-s --K---C'OOSn
alk;.I
prous chloride (1 to 2 grams) suspended in 15 ml. of concentrated hydrochloric acid is drawn into the reaction flask, and the residual cuprous chloride-hydrochloric acid mixture is washed into the apparatus with 30 ml. of water. The flask is heated slowly to boiling during the decomposition, and near the end of the determination the carbon dioxide rate is increased to sweep t'he nitrogen into the nitrometer more rapidly. An analysis requires 30 to 45 minutes. The volume of a blank dctermination, the vapor pressure of 50% potaqsium hydroside solution, Charles's law and Boyle's Ian- are taken into consideration in the calculation. DI scuSSION
Three pure aromatic-substituted triazenes were synthesizetl and analyzed by the procedure outlined. In addition, a sample of commercial triazene was analyzed by this procedure and polarographically. The results are given in Table I. RIono-, di-, aryl-, or alkyl-substituted triazenes react to liberate a quantitative amount of nitrogen. Diazonium compounds can also be determined by this procedure and consequently interfere. A< hYOWLEDGMEVT
The reagents were potassium hydroside solution, 5O%l, 117 weight; cuprous chloride, and concentrated hj-drochloric acid. PROCEDURE
The air is completely removed from the apparatus up t o the reaction flask by displacement with carbon dioxide. h sample t h a t will liberate 15 to 25 ml. of nitrogen is weighed into the reaction flask. The flask is attached to the apparatus, and carbon dioxide is passed through the apparatus until microbubbles arc' seen in the nitrometer. If a slurry or a paste is being analyzed. sufficient water is added so that the delivery tube .V [see drawing. reference (S)] dips just below the liquid level. Carbon diosidcs passing through the solution will displace the dissolved gases. After microbubbles are seen in the nitrometer, a misture of cu-
The author u i s h c ~to eyxess his appreciation t o L. T. Hallett for his encoursge-mnt, t o H. J. Stolten for the polarographic determination. and to L. Frauenfelder and J. Kervenski for some of the experimental work LITERATURE CITED
(1) Houhen-Weyl, "Die Methoden der Organischen Chemie," Tol. IV, p. €168, Stuttgart, Georg Thieme Verlag, 1944. ("'Pierce, d.E., arid Rising, 11. 1f,,J . Am. Chem. SOC.,59, l3fit-2
(1936:. (3) Siggia, Sydney, and Lohr, L. J., ANAL.Crimf.. 21, 1202-3 (lN!H. R E C E I V Efor U review Octoher 3, 1952. Accepted March 17, 1053.
Determination of Caffeine and Trigonelline in Coffee by Paper Chromatography L4WRENCE KOGIN, FREDERICK J. DICARLO, LYD WIYNE E. M4YNhRD T h e E'leischmann Laboratories of Standard Brands, Inc., .Yew Y-ork, Y . P. -HE
successful application of paper chromatography and spec-
7 trophotometry t o the analysis of mixtures of purine and p , rim~
idine derivatives obtained by the hydrolysis of nucleic acids (2-5,10-15) and t o a variety of alkaloids (8) suggested the usefulness of these techniques in the estimation of caffeine in coffee. Pi eliminary experiments with pure caffeine showed that this compound was recovered quantitatively from chromatograms by elution with 0.1 A' hydrochloric acid. Other experiments with mixtures of caffeine and chlorogenic acid [isolated from coffee by the method of Moores, RlcDermott, and Wood ( 7 ) ]demonstrated the advisability of chlorogenic acid removal prior to chromatography. For this purpose, coffee extracts were treated with magnesium oxide ( 1 ) . Wyatt's (IS) solvent mixture containing isopropyl alcohol and hydrochloric acid served well to isolate caffeine on chromatograms prepared from coffee solutiong. Caffeine was located by its fluorescence in ultraviolet light Another coffee constituent which fluoresced upon exposure of the papergrams to ultraviolet light was eluted and identified as trigonelline, a compound investigated chromatographically by Munier and Macheboeuf (9). Chromatographic work on solutions containing caffeine and trigonelline resulted in the quantitative separation and recovery of both compounds. Green and roasted coffee beans, soluble coffee products. and coffee extracts were analyzed for caffeine by the Bailey-ilndrew procedure ( I ) , and for caffeine and trigonelline by the chromatogiaphic method deecribed below. The two methods of caffeine determination yielded values differing by less than 5 q in every instance.
\I aTERI4LS
Sulfuric acid, 0.05 .Y. Magnesium oxide, heavy, U.S.P. Solvent system: 65% isopropyl alcohol, 2.0 S with respect to hydrochloric acid. This should be renewed occasionally as evaporation losses change the proportion of alcohol to acid. Hydrochloric acid, 0.1 AT. Whatman filter paper, KO.1, 6 X 15 inch sheets. Xneralite lamp, Model S o . V-41, The Ultraviolet Products Corp., Los Angeles, Calif. Beckman spectrophotometer, Model DU, equipped with quartz cuvettes. PROCEDURE
Grind roasted or green coffee beans before sampling. Direct11 sample instant coffee or coffee estract. iiccurately weigh sufficient material to give 5 to 10 grams of coffee dry matter and transfer to a tared I-liter Erlenmeyer flask. Bdd 200 ml. of distilled water and 50 ml. of 0.05 X sulfuric acid, and boil the mixture gently for 20 minutes on an electric hot plate. Then add 25 grams of heavy magnesium oxide and boil for an additional 20 minutes. Cool to room temperature, weigh, and add water to replace that lost by evaporation (tare plus sample weight plu. 275 grams). Mix thoroughly and filter through Whatman S o . 12 pap:. L sing a 0.1-ml. graduated pipet, transfer duplicate 0.02-ml. aliquots of the filtrate onto a strip of Whatman S o . 1 filter paper. Place the samples about 3l/2 inches apart on a line drawn 1'12 inches from the bottom edge of the sheet. hfter the paper has dried, add two additional 0.02-ml. portions to the same s otq. allowing the paper to dry betxeen each addition. S u s p e n f t h e paper in a tank containing the solvent pair so that about 1/4 inch of the bottom edge i R immersed. Cover the tank and let stantl
1119
V O L U M E 25, NO. 7, J U L Y 1 9 5 3
350 'O0
eluntes. I n the scheme of trigonellint? :11idysis reported by Moores and Greninger ( 6 ) , a n aqueous alcoholic: coffee estract is passed through a n adsorbent which is later treated with aninioniuni hydroxide to jield a solution containing trigonelline and some foleign niaterial absorbing a t 325 mp. 11oores and Greninger esti~nat,edthe t1,igonelline contents of the eluates in t x o ways. The preferred procedure consisted of rending the absorption :it 265 nip (the absorption niasimum) before :md after the quantitative remov:d of trigonelline by precipit:ition with zinc ferrocyiinide. The other iiiethod involved 2635 nip and a t 325 rending the optical den>ities of the e h t e nip, :ind subtracting the latter iron1 the fornier. IYith estracts from green coffees, the 32.5 mp density readings necessitated cori ~ c ~ t i o of n s about 57;. and gave results which compared favorably \\.it11 those obtained by the ferroryanide procedure. JI-ith estracts from roasted coffees, hoR-ever, the corrections for 325 mp :ibsorption were ahout 30%, and correspondenct* with ferrocy:inide dntn K:W not nlnn>
t ~
22C 230 24C WAVE
250 260 2 - 5 280 2 9 0 3 0 C LENGTH,
mu
Figure 1. Ultraviolet .Ibsorption Curven 011 Caffeine Solutions 10 micrograms/ml. in 0.1 .V hjdrochlorir acid. 1, pure caffeine; 2, eluate from chromatogram prepnred from pure caffeine; 3, eluate of caffeine area from rhromatogram prepared from coffee sample 400
f o r 18 to 24 hour.?. ' r h m remove tlie ~ l i r ~arid e t dry it by esl)omre
to air. Inspect the dry' sheet belore a AIineralite lamp mounted in :1 dark room. l l a h off the lirie reached by the solvent front and encircle fluorescent areas. The cafieine spots will have a n RI.. value of about 0.78 and the trigonelline spots nil1 have a value of about 0.59, (R? is defined as the distance traveled by the spot divicled by the distance traveled by the solvent front.) Cut out the duplicate a r e w antl place each into a test tube. Then cut from the free lane between the fluorescent spots an area of thc same size and RF value and put the clipping into a test tube. Pipet 5 i d . of 0.1 N hydrochloric acid into each tube and let stand at room temperature for a t least 4 houw. Gently snirl the tubes to effect mixing and transfer eluate to quartz cuvettre ior re:ttiing in a Beckman spectrophotometer. Use the eluatv from the free lane as the blank for reading the eluates of thc corresponding fluorescent areas. Using a slit width setting ot 0.74 read the absorbancy of cafieine solutions a t 272 mp and the :ibsorbanry of trigonelline solutions at 285 inp. Calculate results using E:':,:~,,, values of 509 for caffeine and 207 for trigonc~~ine. RESt'LTS \ Y D DISCLSSIOS
The ~)i~ocetlure de,wri\ied was found convenient for the siniult:ineous detrrmination of caffeine :inti trigonelline in coffee s:iniples. The glass tanks used for chromatography were large enough (18 inches high and 10 inches wide) to arconiniodate 8 to 10 sheets each. Thuh, although the elapirtl time of this method involves 2 working days, one oper:itor c:in use sever:il t,:inks antl perform more a n a l y , w in th:it period than is prrmitted by other :inalytical procedurr? for caffeine and trigonelline. T h e solvent teni eniployed Fepnrated the bases effectively; the average RF vnlues were 0.78 for caffeine and 0.59 for trigonelline. Since the compounds fluoi~escetlin ultmviolet light, the need for spraying and washing the paper \ w e obviated. Chlorogenic acid also fluoresced upon exposure to ultrnviolet light, and showed a n K p value of ahout 0.86 in t,he isoprogyl :ilcoliol-hydrochloric acid solvent. Incomplete renioval of c.hlorogenic acid from the coffee estract by magnesium oxide treat,ment did not interfere with the deterniination of caffeine since the chlorogenic acid did not overlap the caffeine spot:: o n the chromatograms. The quantitative separation of chlorogenic acid by the combined techniques of chemical treatment and chromatography circumvented the necrssity of making corrwtions in :ihsorption readings t:ilii.n on trigonelline ant1 caffcint
t
u3
350 300
4 m
233 20 5
153 133 -
\
0531 2 2 2 230 Z L O VrAVE
Figure 2.
250 260 270 280 290 300 -ENSTH
ml U
Ultrabiolet Ahsorption Curves on Trigonelline Solutions
20 micrograma/ml. in 0.1 N hydrochloric acid. 1, pure trigonelline; 2, eluate from chromatogram prepared from pure trigonelline; 3, eluate of trigonelline area trom chromatogram prrparecl from coffee snmple
Table 1.
Caffeine and Trigonelline Yaliies Obtained on Uifr'erent T) pes of Coffee Saniples
Cutfee Santos, green Santos. roasted Colombian, greeil Colonibian. roasted Bukoba, green Bukoba, run-ted Brand .I,roaated Brand B. roasted Brand C, roasted
Soluble D carbohydrate type Soluble E: carbuhydrste type Soluble I:. carlmhydrate tylJe Extract .I Extract B Calculated t o a dry basis.
% Caffeinea ChrouraBaileyt o gra phi c Andreainethod nietiiod
I .09
1.11 1 21 1 15 2.03 2.21
1 . 10 1 . 1.5 1.29 1.24 2 1s 2 .3
.->
41
Trigonelline" 1.01 0 52 0.62 0.57 0 . $52 0 41
1.30 1.17 1.2; 3 44 3 33 3 14
1 31 1 1ti 1 27 3 27 30
3s
1 94 2 ?A 2 ti5
1.60 1.77 1.79
1.39 1.s3 1.80
I 18 0 88 0 so
9.99 4 .iS
3 93 4 3;
2 63 2 89
J
0.80
0 il 0 91
A N A L Y T I C A L CHEMISTRY-
1120
LITERATURE CITED
I n Figure 1 are shown the ultraviolet absorption curves for pure caffeine, a n eluate from a chromatogram prepared from pure caffeine, and a n eluate from a chromatogram prepared from a coffee solution. Figure 2 portrays trigonelline absorption curves obtained in the same manner. The correspondence of these curves illustrated that the caffeine and trigonelline eluates were spectrophotometrically pure. Analytical data are given in Table 1. T h e caffeine results obtained by the new chromatographic procedure agreed well with data yielded by the official method in all cases. Roasting had little effect upon the caffeine content of coffee beans, but resulted in 8 to 49% losses in trigonelline content.
(1) Assoc. Offic. Agr. Chemists, “Official Methods of Analysis,”
7th ed., 14.14, p. 222, 1950. (2) Carter, C. E., J . Am. Chein. SOC.,72, 1466 (1950).
DiCarlo, F. J., Kent, A. M., and Schulte, A. S.,unpublished work. (4) Hotchkiss, R. D., J.Biol. Chem., 175, 315 (1948). (5) Xlarshak, d.,and Vogel, H. J., Federatioil Proc., 9, 85 (1950). (6) Moores, K. G., and Greninger.. D. AI., ASAL. C H E h f . , 23, 327 (3)
(1951).
(7) Moores, R. G., McDelmott, D. L., and Wood. T. R., Ibid., 20,620 (1948). (8) hlunier, R., and Macheboeuf, 31., B d I . soc. ehim. bid., 31, 1144 (1949). (9) Ibid., 33,857 (1951).
(10) Smith, J. D., and Markham, R., Biochem. J . . 46, 509 (1950). (11) Vischer, E., and Chargaff, E., J . Biol. Chem.. 168,781 (1947).
ACKh-OWLEDGMENT
(12) Ibid., 176,703,715 (1948). (13) T y a t t , G. R., Biochena. J . , 4 8 , 5 8 4 (1951).
The authors are indebted to Adrienne XI. Kent and -Anthony Cowia for technical assistance.
R E C E I V Efor D review December 26, 1952.
.iccepted March 11. 1433.
Potentiometric Determination of Boron in Nickel Boride HERMAN BLUMENTHAL
AND WILLIAM FALL American Electro Metal Corp., Yonkers, I?’. Y .
- lloys and borides is generally determined by transthe element into boric acid, separating this acid from the rest of the constituents, and titrating it with sodium hydroxide. This separation is carried out by four basically different methods-distilling the boron off in the form of methyl borate (3),plating the interfering metals out electrolytically ( 2 1 ) or precipitating them ( 1 5 , 7 . 9 ) . and finally with the help of an ion exchange resin (8.8) The senior author ( 1 ) published a method for carrying out this separation by precipitating the interfering metals with barium carbonate. T h e procedure was found effective for a large number of common metals, but it could not be used for the separation of riickel . DETERMINATION OF BORON IN PRESENCE O F NICKEL
Sickel is incompletely precipitated with barium carbonate, even after prolonged boiling. It is believed t h a t this is due to its strong tendency to form complex compounds. Complete precipitation of nickel is not only dependent on the p H of the solution but on many other factors governing this complex-forming tendency. Under the conditions present in this case, nickel was completely precipitated at a pH between 7.2 and 8.8. The p H 7.2, however, is not reached with barium carbonate, and when barium hydroxide or sodium hydroxide were added t o break up any complex formed and to accomplish complete precipitation, varying and considerable amounts of boron were retained by the nickel precipitate, Therefore, the aim was t o find a procedure by uhich boric acid could be titrated in the presence of nickel, Tanabe and Hidaka ( I O ) published a potentiometric titration proredure for boric acid in the presence of a small amount of certain metal ions including nickel, claiming that by adding standardized sodium hydroxide solution from a buret, a complete precipitation of the metal could be achieved before the actual boric acid titration was started. This finding was not verified in this investigation, probably because the nickel content was too high. Hollander and Rieman (6) determined boric acid in glass, and Foote (4)determined boron in Tvaters by a fixed p H titration method. The same procedure was used by Martin and Hayes (8), after removing the metals with a n ion exchange resin. This procedure consists in partly neutralizing the solution of the sample to a more or less arbitrary pH, then adding mannitol to form the strongly acidic mannito-boric acid complex and carrying the titration of this complex only to the original arbitrary pH. In
this type of titration the neutralization of the boric acid is obviously incomplete, and an empirical relationship exists I)et,ween the boric acid and t,he sodium hydroxide solution. This relationship must be established by t,itration of a known amount of boric acid under standardized conditions. Hqllander and Rieman use a fixed p H of 6.3, and realizing the incompleteriess of the boric acid neutralization, make a correction in their calculations. Foote carries the titration to p H 7.6, a t which p H boric acid with no mannitol present is only partially neutralized, while the neutralization of the mannito-boric acid complex is almost complete. The small deviations from the actual neutralization Doints are taken care of in the standardization factor. l l a r t i n and Hayes use the fixed pH 6.9. dssuming the presence of nickel during a fixed pH titration, i t n.ould be partially precipitated when the solution is adjusted t o the fixed p H ; and if equilibrium conditions are reached, the rest of the metal still in solution should not react with the sodium hydroxide during titration. EXPERIMEXTAL WORK
SVith the intention of using the fixed p H method for the determination of boron in the presence of nickel, this method cvas compared TI ith a regular potentiometric titration and a visual titration. For this purpose a mixed indicator n a s added t o a boric acid solution which was then titrated potentiometrically, observing the indicator end points a t the same time. The neutraliza10
9 8 1
I, 6 5 4
0
5
10
15 ML.
PO 25 30 35 OF 0.05 N NaOH
40
45
50
Figure 1. Titration of Boric Acid Solution
55
