The Brown Front in Paper Chromatography ROBERT A. SCHWANE' and GEORGE N. KOWKABANY The Catholic University of America, Washington 77, D. C.
b The brown front in filter paper chromatography and elution is apparently caused by some modification of the filter paper by the wet-dry interface as it travels along the paper. This effect cannot be eliminated by us many as 20 elutions with water. The brown residue is made up of watersoluble organic material and salts, which can modify the chromatographic behavior of sugar acids.
Paper Co., No. X-934-AH) was used in one study. The pieces of filter paper
from which the eluate was collected were 2 cm. x 10 cm. and tapered a t one end. Stability of the Sugar Acids in Solution. The sugar acids can exist in solution in various forms: as free acids, as salts, or as y- and &lactones. A number of authors (2, 4 8) have discussed the relative stability of these forms. From the chromatographic analysis in this research, it was observed that D-glucono-1 4-lactone was converted into D-gluconic acid when it HE FLUORESCENCE so readily dedissolved in water. ~-Glucurono-6,3tected a t the solvent front in paper lactone dissolved in water as the lacchromatography was investigated as tone, but was gradually converted into early as 1934 by Bone (3). Several the free acid a t room temperature. papers on this subject have appeared, After a period of about 3 weeks, there the latest by Schaffcr, Appel, and was an equilibrium mixture of the two Forziati (7))and it has been shown that forms, with the acid form in excess. Effect of Elution on R p When the continuous evaporation of water a t the sugar acids were first chromatoa wet-dry interface on the fibrous graphed (as received from the manumaterial (cotton strips were used) facturers without further purification), resulted in chemical modification of the they showed R f values in the range cellulose (evidence for oxidized polyof 0.1 5 to 0.20. However, when these mer was found) and the formation of chromatographed spots were eluted water-soluble, organic degradation prodand rechromatographed, the R f range ucts. The reaction appears to require was decreased by about one half. oxygen. Ambler and Finney (1) inTable I gives representative R f values for 0.2-mg. saniples of the sugar acids. vestigated the brown front on filter Since the R , values after elution were paper and believe that the mechanism about the same as R , values for the is the formation of a degradation prodqalts of the sugar acids, conversion of uct of cellulose by oxidation a t the wetthe sugar acids to their salts during dry boundary, along with the capillary the elution process seemed probable. concentration of the same or a similar The following experiments were devised product already prescnt in the paper. t o determine the cause of this. This research is an extension of that Two samples of D-gluconic acid, about work into the nature of the brown front 0.2 mg. each. mere spotted on a chronistogram. One of these samples was and its effect on filter paper chromatogoverspotted with the eluate from a filter raphy and the elution process. paper strip, The overspotted sample showed the same R , value as sodium EXPERIMENTAL D-gluconate, whereas the other sample was not retarded. Froin this, it wai Materials and Equipment. The concluded that something from the filter chemicals used in this research were paper was causing the salt formation. D-glucono-1,4-lactone (Fisher, puriIn another experiment, a filter paper fied) and ~-g~ucurono-6,3-lactone strip was eluted continuously with (Eastman, highest purity). All chromatograms were 19 cm. wide and 40 cni. long and developed by the descending method using l-butanolacetic acid-water (4:1:5 v./v.) as the organic phase. The spray reagent used to locate the spots was periodateTable 1. Representative Rf Values of perwangmate (6). The water used for the Sugar Acids elution was triply distilled (once from permanganate). Unless other&e speciBefore After fied, the filter paper was Whatrnan No. dcid e!ution eht.ion 1. Glass-fiber filter paper (Hurlbut 0.17 0.09 D-Gluconic 0.15 0 07 D-Glucuronic 1 Present address, Department of Chemiptry, De Paul University, Chicago 14, Ill.
T
1660
e
ANALYTICAL CHEMISlRY
water, and the successive drops were collected. Each of these drops was overspotted on a separate sample of D-gluconic acid. The salt formation diminished with each successive drop so that, by the eighth and ninth drops, there was little or no conversion of the Augar acid into its salt The brown color of the eluate had also disappeared. However, when the strip was dried and re-eluted, the salt formation caused by the first drop of new eluate was about the same as that caused by the first drop in the initial elution. The color of the eluate was now yellow instead of brown. These results seem to indicate that the brown front formed a t the wet-dry boundary was in some way responsible for the formation of salt from the sugar acid. This material was easily washed out of the paper, the elution occurring a t the solvent front, but when the paper was dried and re-eluted it was formed once more. Experiments with D-glucuronic acid gave the same results. Examination of the Residue from the Eluate. When the eluate from the filter paper was evaporated to dryness, a yellow-brown residue remained. When this residue was heated to combustion in a porcelain spoon the organic material was burned away, but a white salt remained which exhibited a crystalline structure under microscopic examination. There was not enough of the salt for ordinary cheniical analysis. Activation analysis of the ash from Whatman No. 1 filter paper revealed tlie prescnce of sodiuni, but no other element was positively identified by this method. No attempt was made to identify the organic material which was assumed to be degradation products of cellulose. However, to e x a n h e the effect of the organic material on the sugar acids, a drop of the eluate was first treated with Dowex 50-H+ resin to remove cationic material and then overspotted on a sample of D-gluconic acid. The results can be seen in Figure 1, which is a tracing of a chromatogram Sample 3 shows only a little tailing, whereas sample 4, which was untreated by the resin, is definitely retarded (formation of salt of the sugar acid) with a small amount of bearding due perhaps t o the hydrolysis 01" the salt to the sugar acid during chromatographic development. The brown color of the eluate was virtually unchanged by this resin treatment. It may be assumed that the organic material was left in the e!uate. Dowex l, an anion-exchmge resin, removed the broRn color from the eluate, but the
saltrforming property (shown by overspotting technique) remained. Sample 1 of D-gluconic acid was overspotted with water. This was done to show that the salt formation was not caused by something in the water. In another experiment, 1 drop of 10% hydrochloric acid was added to 1 drop of eluate and overspotted on a sample of D-gluconic acid. Very little salt formation was noted, b u ; the strong acid introduced irregularities in the aolvent front. Efforts to Eliminiite the Brown Front. Whatman So. 1 strips were eluted 20 times, with drying between each elution Each elution produced residue containing orgmic material and salt. The amount of residue was small, but the average of about 200 samples was approximately 0.1 mg. of residue per strip per elution. Less than half of this was salt. Other papers were tried, for example, Whatman Nos. 2 and 42. Both of these gave the same type of residue. The Whatman No. 42 (acid washed) paper was eluted 13 times, with drying between each elution. The eluate from each elution was overspotted on separate samples of D-gluconic acid. The effect of converting the D-gluconic acid into its salt was about the same for each elution. F h a t m a n No. 1 paper was washed with 10% hydrochloric acid solution for 24 hours and then washed with water for the same length of time. When this paper was dried and re-eluted with water, residue containing the salt was produced. Glass-Fiber Paper. Glass-fiber filter DaDer should r,ot be affected by th^e wet,-dry inserface. Some strips of this paper were eluted, and a yellow substance appeared to move down the paper, along with the wetdry interface which was rather irregular. When this eluate was evaporated to dryness a residue remained which was made up of salt and organic material. When overspotted on Dgluconic acid, this residue altered the R, value of the acid hi the same way as the residue from cellulose fiber paper. Five or six successive elutions, with drying between each, produced residue, but the yellow front was not noticeable after the second elution. After the sixth elution, the glazs fiber became fluffy and would no lmger tyansport water by elution. This behavior might indicate that there is an organic binder in the glass-fiber paper. The organic material might also have been iutroduced during the maniifacturing process. For example, sometimes the glass fibers are beaten in 8 citric acid solution to give a good dispersion. DISCUSSION
As others have shorn, the conditions that prevail a t the wt--dry iiitrrfnce which travels along the filtcr pspt'r in the elution of a chroniatogram c:iiise wnie degradation of thl: paper to take 1,lac.e. This is evidented in part by
the production of a small amount of water-soluble material which comes from the paper, concentrates in the solvent front, and, on elution and evaporation, gives a brown residue. It is now found that, in the presence of this material, the chromatographic behavior of eluted sugar acids is altered. The changes in Rf values can be attributed to neutralization of the acids or to a salting-out effect (6). The change in R f values appears to he due to inorganic cations, a component of the eluate that has not been recognized previously. This cationic material may be hydrolyzed during chromatography, with formation of salts of the sugar acids. On combustion, the brown residue is converted t o a white, crystalline,
I a lCRf TT
=.I7
3Jmm I 1
't a -SOLVENT
ORIGIN
j
/+Rf
Figure 1. gram
FLOW 4
z.09
(9)
D-Glwconic acid chromato-
1. Overspotfed with water 2. No overspotting 3. Overspotted with eluate treated Dowex 50 4. Overspotted with untreated eluate
in this simple fashion. Third, water alone is capable of effecting the release of the cationic material. The key condition appears to be the presence of a wet-dry interface on the paper. Observations can support only an explanation that relates the oxidative degradation a t the interface to an enhanced liberation of cationic impiirities. On the one hand, the cations behave as though they are located in inaccessible (crystalline) regions of the cellulose where protons from mineral acid are unable t o replace them. On the olher hand, the degradative process a t the interface is able to effect replace ment of the cations by protons from the relatively weak acids resulting from the degradation. In the latter situation, only a portion of the cations associated with the paper is thus removed, and drying the paper and re-eluting liberate more. The eluted material is not basic; however, it is able to effect this neutralization. More surprisingIy, overspotting with comparable amounts of sodium chloride was found to produce similar lowering of the R, values of the sugar acids. This is clearly related to the experiments of Westall, who has shown that, during chromatography in a 1butanol-acetic acid-water developer, sodium chloride hydrolyzes with separation of the sodium and chloride ions
with
salt-like material which also has the ability to change the Rf values of the sugar acids. As would be expected if they contained cationic materials, the brown and white materials lose their ability to affect the acids upon treatment with Dowex 50-H+. Some inorganic, cationic material is present in filter paper, probably in association with the carboxyl groups that cellulose is known to possess. Passage of molecules of an aldonic acid in the vicinity of a cellulose-carhoxuglate salt residue would be expected to result in a transfer of the cations by mass-action effects with the resulting formation of aldonate salt. However at. tractive, this explanation is inadequate for three reasons. First, this process should occur a3 well during chromatographic development of the sugar acid on paper before elution, but it does not to any significant extent. Second, extensive washing of the paper with dilute hydrochloric acid should be more eflective in replacing the cations held
Apparently, long exposure t o air makes the paper more siisceptible to modification by' the wet-dry interface. It was noted in this work that the first elution of a paper generally produced the brownest residue, but n9t necessarily the most salt. During successive elutions, the bromn color changed to yellow and finally to a light cream color. If a paper Was exposed to air for a period of 2 weeks or more between elutions, the color of the residue from the elution following this period was a bit darker than the residue from the previous elution. In regard to chromatography, i t does not, seem practical to wash the filter paper repeatedly in an effort to get rid of the residue from the brown front, since 20 elutions were not S I N cessful. To minimize the amount of residue, it seems best to elute the paper once with water shortly before use in chromatoeraphv. If one is attempting to purify B substance by chromatography, it seems best to purify as large a sample as possible on one chromatogram, within limits, to minimize the relative amount of residue from the brown front; or to remove saltq, the eluate, containing a siigar acid or other organic acid, could be treated with a cation-exchange resin in the acid form. Further work in this area is currently in progress. VOL 35, NO. 11,
OCTOBER 1963
1661
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. The 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. The 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
