LITERATURE CITED
(1) Ambler, H. R., Finney, C. F., Nuture 179, 1141 (1957). (2) Bates, F. J. et al., “Polarimetry,
Saccharimetry, and the Sugars,” N.B.S. Circular C440, National Bureau of Standards, Washington, D. C., 1942. (3) Bone, J. W., J. SOC.Dyers Colourists 50, 307 (1934).
(4) . . Hirsch. P., Rec. Trav. Chim. 71. 999
(1952).
’
’
(5) Knight, C. S., Chromatog. Rev. 4 , 67 f 1962). ( 6 ) Lemieux, R. U., Bauer, H. F., ANAL. CHEY.26, 920 (1954).
(7) Schaffer, R., Bppel, VV. D., Forziati, F. H., J. Res. Nail. Bur. Std. 54, 103 (1953). (8) Smith, F., J . Chem. Soc. 1944, 584.
Determination of Nicotinic Paper Chromatography
19) . , Westall, R. G.. Biochem. J . 42. 251 (1948). Partridge, S. M., Biochem. SOC. Symp. Cambridge, Engl. 3, 54 i 1950). ,
I
RECEIVED for review September 21, 1962. Accepted May 15, 1963. Division of rlnalytical Chemistry, 145th Meeting, ACS, New York, N. Y., September 1963.
Acid in Coffee by
HELGA BODDEKER and A. R. MlSHKlN Westreco, Inc., Marysville, Ohio
b A new method has been developed for the determination of nicotinic acid in coffee. A mild acid treatment of the coffee solution or suspension was followed by neutralization and decolorization with basic lead carbonate. After filtration, the supernatant was resolved by paper chromatography, and the nicotinic acid spots were revealed by a cyanogen bromidebenzidine reagent. The density of the spots was determined with a photoelectric densitometer, and the concentrations were calculated through the use of a standard curve. A study was made of the nicotinic acid content as related to the degree of roast.
M
presently used for the quantitative determination of nicotinic acid are based upon biological procedures which are lengthy and require specialized equipment. Chemical methods which have been applied are complex and unsuitable for the determination in coffee. The most common microbiological methods for the quantitative determination of nicotinic acid are those used by Teply, Krehl, and Elvehjem ( I C ) , Bressani and Navarrete ( d ) , and Cravioto, Guzman, and Suarez (3). A bioassay method has been based on the growth rates of rats ( I S ) , and a colorimetric method based on the Konig color reaction has been used by Gassman and Ehrt (6) and by Adamo ( I ) . This color reaction was investigated thoroughly by Gassman (4)and OST METHODS
Type of coffee
Instant Roast
1662
applied to the analysis of foods. However, the method is complex, requiring three corrections for blanks, and the relative standard deviation is approximately &lo’%. Kuznetsora and Chagovets (IO) used a paper chromatographic separation of nicotinic acid, followed by a colorimetric determination in the eluate from the paper using a color reaction with methyl-p-aminophenol. A reliable and rapid method for nicotinic acid analysis nould be of distinct advantage, especially in the food industry. K i t h modifications of a method for the determination of caffeine and trigonelline in coffee ( 9 ) and with use of a solvent system suitable for the separation of nicotinic acid ( 7 ) ,a method of quantitative paper chromatography similar t o that described by McFarren, Brand, and Rutkowski (11) has been developed. This method is rapid and reliable and appears to be adaptable t o the analysis of various food products. EXPERIMENTAL
Procedure. T h e roasted coffee beans were ground t o a fine pon-der. When soluble coffee powders were used, no special preparation was required. A 10.00-gram sample of coffee was mixed with 110 ml. of l . l X hydrochloric acid. The sample was refluxed for one hour and allowed t o cool t o room temperature. T h e acid was neutralized with 40 grams of basic lead carbonate powder
[Pb(C03)rPb(OH)2
Table I. Nicotinic Acid Recovery Original nicotinic acid Ncotinic Calculated Amount content found, acid added, total, found, mg./100 g. mg. mg./100 g. mg./100 g. 32.2 15.0 46.7 47.2 13.4 15.0 27.5 28.4
ANALYTICAL CHEMISTRY
Recovery, % 98.8 97.0
Matheson, Coleman & Bell Chemical Co.]. The mixture was transferred t o a 200-ml. volumetric flask and diluted to volume. -4n aliquot was filtered, and the filtrate was used for analysis. The filtrate was spotted with a micrometer syringe (Burroughs Wellcome Co., London, England) on Vhatman KO. 3MR4 chromatography paper in amounts of 0.02 ml., 0.04 ml., and 0.06 ml. for instant powder samples, and 0.05 ml., 0.10 ml., and 0.15 ml. for roast coffee samples. Five spots of a nicotinic acid solution, prepared from sublimed and dried nicotinic acid, were applied to the same chromatoqam in amounts ranging from 0.3 to 1.5 kg. per spot. The papers were allon ed t o develop by descending chromatography for 16 hours using 1-butanol-ammonium hydroxide-mter (100:2: 16) as a solvent qystein. The chromatograms were dried for 30 minutes in a stream of warm air. The dried papers were placed for 2 hours in a closed tank containing cyanogen bromide vapors (fume cupboard). These vapors were produced by placing cyanogen bromide crystals in t h e bottom of the chamber. After the cyanogen bromide treatment, each paper mas sprayed with a solution of 0.25% benzidine in 50% ethanol and allowed to dry in air for 30 minutes. The preparation of fresh spray reagent each day was necessary. The nicotinic acid ( R , = 0.13) appeared as an intense pink spot on a white background. The masimum density of the spot was determined with a Photovolt electronic densitometer, Model 525 (Photovolt Gorp., 95 Madison Avenue, Kew York 16, E. Y.) using a green filter and a slit width of 6 mm. The instrument was first standardized on a blank portion of the chromatogram, and then the maximum density of each nicotinic acid spot was determined by moving each spot slowly over the slit of incident light until maximum deflection was obtained on the galvanometer. The densities of spots of known concentration were plotted on semilogarithmic paper with concentration as ordinate on the
0
I
I
5
IO
%
wmnr Losr
1
I5
I
PO
I
25
I
30
ON ROASTING
Figure 1. Relation between nicotinic acid content of coffee and degrfee of roast
log scale and density :is abscissa. From these standard curve: and the recorded densities, the concentrations of the experimental samples were calrulated. Recovery Experiments. Neasured amounts of resublimed nicotinic acid were added t o roast ( offee and instant coffee samples. T h w e samples were analyzed by t h e described procedure, and the recovery was calculated. The theoretical total was determined by adding t h e quantity which had previously been found in the coffee samples to the amount which was added. The recovery data are given in Table I. T h e Change in the Nicotinic Acid Content with Degree of Roast. A series of samples of Arabica and Robusta coffees mas roasted t o varying degrees. Each s a m d e consisted of 300 grams of unroasxd coffee beans. After roasting, the sample was weighed to determini? the weight lost on roasting. The nicotinic acid cont e n t of these coffee samples of different roasts was deiermined as outlined previously. This data are plotted in Figure 1. RESULTS A N D D SCUSSION
The preparation of the sample was important in obtaining a reliable nicotinic acid analysis. When roast coffee samples were used, the reaction medium was heterogeneous since the roast coffee remained largely insoluble; therefore, assurance of the complete extraction of the desircd component was necessary. Pre1imin:try experiments showed that grinding the roast beans to a fine powder ensured complete extraction. Hydrolysis wil h dilute hydrochloric acid was introduced to free the
nicotinic acid from any chemical complexes. The neutralization of the hydrochloric acid v, ith lead carbonate resulted in a precipitate of lead chloride which removed most of the dark coffee color. The light brown filtrate was suitable for paper chromatographic reqolution. Exactly 40 grams of lead carbonate was found by experimentation to result in best recovery of nicotinic acid. Therefore, this amount wa3 used for neutralization of each sample to ensure that there mere no variations in analytical results due to different amounts of insoluble material in the T olumetric flasks. The recovery experiments showed that the lead chloride precipitate did not remove any of the nicotinic acid. They also indicated an adequate degree of reproducibility. The precision of the method was determined by analyzing the nicotinic acid content of an instant coffee powder. The average of bix determinations was 25.6 mg. per 100 grams of coffee. The standard deviation wa5 = t ~1.05 mg. Green coffee has been reported to contain very little nicotinic acid. Commercial green beans hare been processed by drying to stop enzyme action. By a microbiological assay, Bresani and Xararrete ( 2 ) found 0.6 to 1.3 mg. of nicotinic acid per 100 gramq of green coffee The cyanogen bromide-benzidine test did not indicate any nicotinic acid in green coffee. The more sensitive p-aminobenzoic acid qpray reagent (8) ihowed a faint trace of nicotinic acid. However, the amount prc-ent was too .mall to be quantitatively determined. Several researchers (1-3) report that the nicotinic acid content of the coffee increases with darker roasts. Trigonel-
line is converted to nicotinic acid by removal of the methyl group from the nitrogen of the pyridine ring. Since the trigonelline content of coffee has been shown to decrea\e during roasting ( I d ) , this convercion is assumed to be the source of the increased nicotinic acid content. This assumption is promoted by the work of Hughes and Smith (6) iTho heated trigonelline in a sealed tube and produced nicotinic acid. Therefore, u-ing the analytical technique described, a d u d y was undertaken to determine the nicotinic acid content in coffee samples with increasing degrees of road. Two varieties of coffee beans were u-ed, Robusta and Arabica. For comparative purpose5 the data from both these varieties were plotted on the same graph (Figure 1). The data from the two types of coffee are very similar, and a single curve is sufficient to represent both varieties. The rate of nicotinic acid formation varies with the degree of roast. Several instant coffee powders were analyzed, and the nicotinic acid content was in the range of 18 to 40 mg. per 100 grams of instant coffee. This is in accordance with the data reported by Teply (IS) and by Hughes and Smith (6). ACKNOWLEDGMENT
The authors gratefully acknowledge the help of M. L. ~~701from, The Ohio State University, in the preparation of the manuscript. LITERATURE CITED
(1) Adamo, G., Boll. SOC. Itul. Bid. Sper. 31, 79 (1955) ( 2 ) Bressani, R., Navarrete, D. A., Food Res. 24, 344 (1959). 13) Cravioto, R. 0 , Gu~man, G. J., Suarez, RI. L., Cienciu Mer. 15, 24 (19.55). (4) Gassman, B.,Nahrung 4, 149 (1960). (5) Gassman, B., Ehrt, D , Emoehrungsforschung 4, 72 (1959). (6) Hughes, E. B., Smith, R. F., Sac. Chern. I d . 65,284(1946). ( 7 ) Kende, H., Ber. Schweiz. Bofan. Ges. 70,232 (1960). (8) Kodicek, E., Reddi, K. IC., Sature 168, 475 (1951). (9) Kogan, L., Dicarlo, F. J , Maynard, '8. E , ANAL. CHEV. 2 5 , 1118 (1953). (IO) Kuznetsova, C. I f . , Chagovets, R V., Ckr. Biookhim. Zh 27, 187 (1955). (11) McFarren, E. F., Brand. Kathleen, Rutkowski, H. R , ANAL CHEV. 23, 1146 (1951). (12) Slotta, K. H., Pl'eisser, K , Ber. 72B, 133 (1939). (13) Teplv, L J., Food Technol. 12, 485 (1958). (14) Teply, L J , Krehl, W -4, Elvehjem, C. A,, Arch Rzocherii. Biophus. 6 , 139 (1945)
RECEIVEDfor review April 8, 1963 Accepted July 12, 1963. Westreco, Inc., is a subsidiary of the Jnternational Seat16 Co.
VOL. 35, NO. 11, OCTOBER 1963
1663.
