ULTRAVIOLET ABSORPTION TECHNIQUE TO DETERMINE^

194 FLRIDA STATE HRTICULTURAL SCIETY, 1958 Effect of Different Abuse Treatments. ther processing conditions that would influ ence the ultraviolet absorption spectrum of grapefruit juice were investigated. A grape fruit juice was abused by aerating it for five minutes with a high speed stirrer, a second portion was partially evaporated and pure water replaced the evaporated portion, while a third portion was allowed to stand at room temperature for 20 hours. Figure 4 presents the results of these abusive treatments and shows, by increased absorption, the respective degree of abuse. The increase in a juice's ab sorption would therefore seem proportional to the degree of abuse. Severe abuse appeared to jeopardize the accuracy of this ultraviolet technique for measuring naringin content; however, no processor could afford to abuse a juice to this extent. Effect of Temperature.-Finally, an experi ment was designed to show the absorption changes that occurred when a raw grapefruit juice was heat pasteurized at various tempera tures. Approximately five ml. of juice in a spiral glass tube was immersed for threefourths of a minute in a water bath held at 180, 200, and 212 F. in consecutive ex periments. The spiral tube with the juice sample was immediately reimmersed in a cold water bath until the juice returned to room temperatures. Figure 5 shows the results of these heat treatments. Most evident was the increased absorption at higher processing tem peratures. The disappearance of a shoulder at 325 mu. was again found to indicate severe abuse. Summary and Conclusion A new ultraviolet technique has been de scribed for analyzing the effect of several adverse factors upon grapefruit juice. Vari ations in processing conditions brought about proportional ultraviolet absorption changes in the 260 to 360 millimicron range. Among the processing factors that were found to influence the ultraviolet absorption of grapefruit juice were extractor pressure, degree of heat stabili zation, peel extractives, rag and pulp addi tions, length of storage at room temperatures and aeration. The procedure appeared to make available, for the first time, a method whereby qualityconscious processors can measure the effect of processing factors that contribute to flavor deterioration in grapefruit juices. LITERATURE CITED 1. Hendrickson, R., J. W. Kesterson and G. J. Edwards. Ultraviolet absorption technique to determine the naringin content of grapefruit juice. Proc. Fla. State Hort. Soc. 71:. T958. 2. Wenzel, F. W., E. L. Moore and R. W. lsen. Fac tors affecting the flavor of frozen concentrated orange juice. Program of Eighth Annual Citrus Processors' Meeting, Citrus Experiment Station, Lake Alfred. Sept. 26, 1957. ULTRAVILET ABSRPTIN TECHNIQUE T DETERMINE^ THE NARINGIN CNTENT F GRAPEFRUIT JUICE R. Hendrickson, J. W. Kesterson, and G. J. Edwards Florida Citrus Experiment Station Lake Alfred The need for a more specific method to analyze for naringin has been apparent for some time because taste panels have not been able to correlate the bitterness of grapefruit juice with the naringin content as determined by present methods. This lack of correlation may be caused by the effect of acidity on bitter taste of naringin, but it might also be Florida Agricultural Experiment Station Journal Series, No. 796. caused by variations in quantity of materials interfering with the chemical determination at certain periods of the processing season. The present method of analyzing for naringin, the Davis test (2), is rather non-specific, de pending on the quantity of yellow color form ed in the presence of alkaline diethyleneglycol. Ascorbic acid produces five to six percent of the total color developed by grapefruit juices (2), but the Davis test avoids interference from many of the other natural constituents of grapefruit juice by virtue of the high sen sitivity of the procedure to naringin. It is possible that the development of a more spe cific method for analyzing naringin would only substantiate the reliability of the Davis

HENDRICKSN ET AL: NARINGIN 195 method. n the other hand, a more discrimi nating method might establish a criterion by which quality and maturity of grapefruit could be evaluated. A new, more sensitive ultraviolet method of analyzing for naringin content of grapefruit juice has been found by the authors. The pro cedure depends on the peak ultraviolet ab sorption of naringin at 285 mu. wavelength which was investigated in a recent examina tion of bioflavanoid analytical techniques (3). Procedure Approximately 25 ml. of grapefruit juice is adjusted to a ph of 3.5 ± 0.1 by the addition of one or two drops of concentrated hydro chloric acid when necessary. This sample is diluted 20:1 with commercial 99 percent isopropanol by pipetting 0.5 ml. of the juice into a 10 ml. volumetric flask and diluting to the mark with alcohol. The alcohol insoluble solids of the juice are precipitated and slowly coagulated. After two hours, a sparkling clear sample is obtained by filtering off the alcohol insolubles using vacuum and a Whatman No. 42 paper disc in a perforated gooch. 1.9 1.7 (7) z y 0.9.5 DILUTIN EFFECT 1/4 ML JUICE 300 20 40 Mu &f (7) z CL 0.7 \ 1 H i / / SLVENT DMF. EFFECT s isoprpyl Alcohol \ WATER w V \ 60 80 300 20 Fig. ]. A comparison of the ultraviolet absorption of grapefruit juices diluted with various solvents. Fig. 2. Effect of grapefruit juice dilution on its ultra violet absorption. The clear diluted solution is evaluated for naringin content by placing a portion of the solution in a 10 mm. silica cell, which is placed in a Beckman DU Quartz spectrophotometer with a hydrogen lamp. The instrument is ad justed to have a 0.15 mm. slit and sensitivity varied to give 100 percent transmission at 360 mu. for 99 percent isopropanol. ptical density readings are taken at wavelengths of 290 and 295 millimicrons and the quantity of naringin interpolated from the difference between the two readings. The approximate percentage of naringin is equivalent to this difference divided by four, but is more ac curately determined with a standard curve. Experimental Results and Discussion Effect of Solvent* Since the intensity and position of an absorption band of a compound can vary with the solvent, three different solvents were tested. In Fig. 1 is shown the 4oo ultraviolet absorption obtained from 240 to 400 millimicrons, when an average single strength grapefruit juice having 0.047 per cent naringin was diluted in similar fashion

196 FLRIDA STATE HRTICULTURAL SCIETY, 1958 2.5 2.3 1.9 1.7 >. 1.5 1.3 _l 5 i.i Q. 0.9 0.7 0.5 0.3 60 P 300 20 40 60 80 Fig. 3. Variations in the ultraviolet absorption of grape fruit juice brought about by ph change. with water, isopropyl alcohol and dimethyl formamide. With water as the diluting sol vent, no absorption peaks were formed, but only shoulders with a lower absorption over the critical wavelengths. Dimethyl formamide gave equally pronounced absorption peaks as isopropanol and its higher boiling point was considered an advantage which led to its ini tial choice. However, it had a sharp cutoff (high solvent absorption) at 260 millimicrons and below, and was finally discontinued be cause the solvent gave variable absorption peaks with age. Effect of Dilution. The optimum concen tration of juice in isopropanol was evaluated by diluting various quantities of grapefruit juice with alcohol. The results (Fig. 2) estab lished that 0.5 ml. of juice diluted to 10 ml. with isopropanol was the best dilution. At a greater dilution samples of low naringin concentration, such as the one shown (0.024 percent), did not have as distinct a peak. This was felt to be a disadvantage even though optical density readings in this range are usually more accurate. At the lowest di lution there appeared to be some depression of the 285 mu. peak with the further disad vantage of operating in the more inaccurate portion of the optical density scale. Effect of ph. In an attempt to explain some of the variations that had occurred in the early phases of this investigation, the ef fect of sample ph was investigated. Some of these results are shown in Fig. 3. As ph was increased the degree of absorption at 325 mu. consistently decreased. Peak absorption at 285 mu. decreased as ph was increased until ph 6.1 was reached. Above this ph the trend was toward a higher absorption value. Synthetic juices of citric acid and alkali with and without naringin showed naringin to have a maximum 285 mu. absorption at a ph 4.9, but this was influenced somewhat by the citrate absorption which also varied with ph. At the lower ph the citrate absorp tion, which also varied with ph, was smaller and varied less over the wavelength range investigated; therefore ph 3.5 was adopted as part of the standard procedure. Effect of Numerous Juice Constituents. f importance to this new naringin analytical procedure was an understanding of how oth er constituents in grapefruit juice contributed to its ultraviolet absorption. In Fig. 4 the indi vidual and additive absorption effect of many of the predominant constituents of grapefruit juice are shown. In these experiments the chemicals were added in proportion to the concentrations normally expected to be found in grapefruit juice. The amino acids included were aspartic acid, asparagine, serine, alanine, arginine, and proleine. Curve 2 shows the in creased absorption contributed by adding suc rose, dextrose, citric acid, inositol, and potas sium dihydrogen phosphate to the amino acid solution. The addition of ascorbic acid re sulted in a pronounced absorption below 270 mu. with its peak value occurring at 245 mu. A small increased absorption, occurring mostly at 320 mu. is contributed by grapefruit oil at the normal concentration level. Curve 5, representing a composite. solution of all the compounds mentioned above with 0.05 per cent naringin, is an attempt to make a syn thetic grapefruit juice. This curve closely re sembles Curve 6, an absorption curve of a commercial grapefruit concentrate. The syn-

HENDRICKSN ET AL: NARINGIN 197 EFFECT F JUICE CNSTITUENTS AMIN ACIDS (D + SUGARS, CITRIC ACID INSITL ft MINERALS (D + + ASCRBIC ACID 0 + + @ + 6FT- L + + + (f GRAPEFRUIT JUICE 80 300 20 40 Fig. 4. A comparison of the ultraviolet absorption con tributed by some of the constituents of grapefruit juice. thetic juice was calculated to have naringin content equivalent to the grapefruit juice shown in Curve 6. Effect of Naringin Concentration. The ef fects of adding known quantities of naringin to a commercial grapefruit juice and similarly the result of subtracting a known amount of naringin are shown in Fig. 5. The latter sub traction was accomplished by allowing the blank to contain a known quantity of naringin, which in this case was equivalent to the Davis analysis of the grapefruit sample. Large changes in peak absorption at 285 mu. with small changes in naringin concentration are to be noted in these absorption curves. ther alternative procedures for measuring naringin content from a juice's ultraviolet absorption curve were an optical density dif ference test and a base line technique (I). However, none of these procedures were as accurate a measure of naringin content. In one case an attempt was made to correlate naringin concentration with the difference in optical density at 285 mu. and 325 mu., a wavelength at which naringin has a secondary peak. The peak at 325 mu., however, was too Mu 80 largely influenced by concentration of other materials in grapefruit juice. In the base line technique a line AB was drawn tangentially through the minimums on either side of the main peak as shown in Fig. 2. The length of the vertical line CD, drawn between the tan gential line and juice's spectral curve, was to be an index of concentration. Variation in vitamin C content was found to influence ap parent naringin concentration by this method. The ultraviolet technique for measuring naringin content in grapefruit juice that was finally adopted is essentially a first derivative technique. The procedure was derived by plotting the change in naringin absorption per five mu. change in wavelength, from which maximum absorption change was found to occur at 295 mu. The difference in optical density between 290 and 295 mu. was con sequently used as a measure of naringin con centration and in this narrow wavelength range was found to be less affected by ab sorbing impurities than the other techniques evaluated. Analysis of Various Grapefruit Juices. The procedure described initially was found capa- (0 8 2.1 r 4.7 1.3 S 0.9 Q. 0.7 0.3.I CNCENTRATIN EFFECT GRAPEFRUIT JUICE (5) + al% NARINGIN + a2% + 0.03% " - 0.04*4 " 40 60 80 300 20 40 60 80 Mu %fg Fig. 5. Influence of small increments of naringin on the ultraviolet absorption of grapefruit juice.

198 FLRIDA STATE HRTICULTURAL SCIETY, 1958 Table 1.- A comparison of analyses of grapefruit juices by the ultraviolet absorption technique and Davis test. Sample Extraction Pressure UV technique Percent Naringin Davis test L09A4 L09B4 L09C4 0.036 0.051 0.075 0.038 0.054 0.070 B15B5 B15C5 B15A5 0.061 0.061 D20B5 D20A5 D20C5 0.053 0.059 0.068 0.050 0.066 F14B5 F14A5 F14C5 0.055 0.064 0.046 0.050 ble of determining naringin concentration ac curately in pure solution or added increments to a grapefruit juice. In analyzing pure grape fruit juice, however, the problem was different in that there was no juice available with a known naringin content or an absolute stan dard for comparison. The naringin analyses of grapefruit juices by the ultraviolet absorp tion technique in Table 1 were therefore com pared against Davis test results which were assumed to be reasonably accurate. The grape fruit juices in the study were processed at four stages of maturity and extracted in the pilot plant at the Citrus Experiment Station by a citromat at three different pressures. The first three samples were processed in December, the second group in February, the third in April, and the fourth in June. Within each group the samples were placed in order of extraction pressure used, that is, low, medium, and high. A comparison of results by the ultraviolet method versus the Davis test showed the former to yield lower answers at the start of season and successively higher results with increased maturity and pressure. A similar conformity was noted in the analysis of com mercial grapefruit concentrate. The procedure also appeared capable of measuring degree of abusive processing conditions simultaneously and will be described in another paper. Summary and Conclusions A new ultraviolet method for determining the naringin content of grapefruit juices was described. The important conditions have been established and described for consistent results with this method and comparable analyses given for grapefruit juices by the ultraviolet technique and the Davis test. In the absence of an absolute standard, the ul traviolet results would appear to substantiate that the Davis test does not read high with mature or over-mature samples of grapefruit juice. LITERATURE CITED 1. Biffen, F. M. and W. Seaman. Modern instruments in chemical analysis. 333 pp. 1956. McGraw-Hill Book Co., Inc. 2. Davis, W. B. Determination of flavanones in citrus fruits. Anal. Chem. 19: 476-8. 1947. 3. Hendrickson, R. and J. W. Kesterson. Chemical analy sis of citrus bioflavanoids. Proc. Fla. State Hort. Sor 70: 196-203. 1957.