CoIo rimet ric Dete rmination of Hy d roxy proIine D. S. M I Y A D A and A. L. TAPPEL Department of Food Technology, University o f California, Davis, Calif.
The absorbance a t 560 mp is a linear function of the hydroxyproline concentration within the range of l to 15 y per ml. H o n ever, at higher concentrations this relationship does not hold.
Some of the principal factors influencing the colorimetric determination of hydroxyproline were studied. .in improved procedure and its application to the determination of hydrouj proline in gelatin and collagen are given.
RESULTS AND DISCUSSION
B
ECAUSE of the uniquely high content of hydroxyproline in collagen, colorimetric determinations of hydroxyproline have been employed almost exclusively in the analysis of collagen in the connective tissue of muscle. Since the colorimetric method \\-as first introduced b>- Seuman and Logan in 1950 (3),valuable coritri1)utions have been made by Baker, Lampitt, and Bron-ti ( 2 ) and Kierbicki and Deatherage (5) on factors affecting the awuracy of the method. Nevertheless, the factors specifically as.miated with the processes of color development have never heel] reported. Iluring the course of analysis of beef muscle for collagen and e l u t i n by the Neuman and Logan method, instability of the color was found to limit the precision of the measurements. T o obtain hetter precision, a study was made of the factors associated w i t h color development. On the basis of the results obtained, a modification of the Xeuman and Logan method was made. Tliih modified method was applied to the analysis of available samplcs of collagen arid gelatin and the results are conipnred with other values found in the literature.
The effects of sulfuric acid concentration and time on the color produced x i t h 30 y per ml. of hydroxyproline solut,ion xvere studied liy recording the spectra in the region 450 to 700 mp (Table 11. In all cases, the maximum absorbance occurred a t 560 nip. This study also shows that the color is more intense in 3.0.1. than in 1.5.V or 9.0X sulfuric acid, whereas the color fades more rapidly a t high hydrogen ion concentrations. The effect of hydrogen ion concentration on color stability vias determined by adjusting the p H after the color was developed and observing the change in absorbance as a function of time (Table 11). In this esperiment, a precipitate formed in the sample buffered a t neutrality. The precipitate was removed by filtration and the absorbance readings reported iTere taken on the filtt,ate.
200
RLATERILS 4YD EQUIP3IENT
Copper sulfate, 0.01M. Podium hydroxide, 2.00X. Hydrogen peroxide, 6%. Sulfuric acid, 1.5, 3.0, and 9.0A\-. -4niino acids (tryosine, tryptophan, and hydrosyproline) xere ohtnined from the Sutritional Biochemicals Corp. and made into solutions of required concentrations. p-Dimethylaminobenzaldehyde (Eastman grade organic chemical) was recrystallized twice from ethyl alcohol according to the dii.ections of Seuman and Logan ( 3 ) . 1-Propanol n-as also an Eastman grade organic chemical, and the fraction distilling between 96" and 97" C. was used. The 5 o/o p-dimethylaminobenzaldehydereagent \vas made b>dissolving 5 grams of p-dimethylaminobenzaldehyde in l-propnnol and diluting to 100 ml. Collagen samples n-ere obtained from cattle hide (4)and from the distal portion of the deep and superficial flexor tendons of the fore and hind legs of cattle. Tendon collagen was prepared according to a procedure described by Baker, Lampitt, and Br,ou-n (2). Gelatin samples were products of Nutritional Biochemicals Coqi. and Charles B. Knos Gelatin Co., Inc. J l o s t absorption spectra and ahsohanee values were obtained with either a Beckman Model D R spectrophotometer or a Beckm x i Jlodel D U spectrophotometer. The values in Figure 1 \$-?re obtained with a Xlett-Summerson photoelectric colorimeter.
0 1 IC
909
I
30
I
I
I
40
50
60
MINUTES
Table I.
Absorbance of color as a function of heating time at two different temperatures
Effects of Time and Sulfuric k i d Concentration on Absorbance of Colora
Xormality of Sulfuric Acid Added 1 5b 3 0 9.0 0.290 0,492 0,091 0.326 0.414 0.070 4 0,342 0.352 0.060 20 0.294 0 096 0.023 R r a d i n v obtained a t 560 m p . Final hydrogen ion concentration is 0 . 3 3 4 s . Time. Hour,
;
a 6
ml. of 6 % hydrogen peroxide. The resulting solution is shaken first on a rotator for 5 minutes and then in an 80" C. water bath for another 5 minutes. The solution is then cooled in ice 11-ater. For samples of relatively l o x hydroxyproline content, it is recommended that 0.05M cupric sulfate be used ( 1 ). After cooling, 4 ml. of 1.5N sulfuric acid are added with shaking, followed by 2 ml. of 5 % p-dimethylaminobenzaldehyde in 1-propanol. The solution is shaken for 3 minutes and then placed in :II~ 80" C. water bath for 30 minutes to develop the color. The alworbance of the resulting color is read a t 560 mp. A4blank is used in which 1 ml. of r a t e r is substituted for the sample.
I
20
Figure 1.
REC03IMENDED PROCEDURE
T o 1 ml. of sample or standard solution containing from 1 to 1; y of hydrosyproline are added the following reagents: 1 nil. of 0.01M cupric sulfate, 1 ml. of 2.00Y sodium hydroxide, and 1
'
" O 0 I
Table 11.
Effect of Hydrogen Ion Concentration on Absorbance of Colora
Final Hldrogen Ion Concentration 0 0OO.Y (PH 7 0 ) 0 223\0 6222 0 265 0 , 26.5 0 0.250 60 0.24" 0.268 0.247 120 0.236 0.264 0.220 0,253 0.197 180 0.229 Readings obtained a t 560 ~ n p . Time lllnute,
ANALYTICAL CHEMISTRY
910 Table 111.
€I>-droxyprolineContent of Some Gelatin and Collagen Samples Hydroxyproline,
Sample Tendon collagen Cattle hide collagen Gelatin (Knoxj Gelatin ( S B C ) Gelatin Gelatin (Bacto-Difco) Connective tissue Connective tissue Cattle hide gelatin
Sitrogen,
“0
“0
14.2 14.3 15.3 14.3 1 4 . 4 2 f5) 13.6 (8)
16.58 17.25 17.21 17.66
1 2 . 3 s is) 13 2 ( 9 ) 13 2 is)
, . .
...
...
... ...
Table IV. Characteristics of Absorption Spectra Obtained w-ith Tyrosine, Tryptophan, and Hydroxyproline
lil.ir
Amino Acids Tyrosine Tryptophan Hydroxyproline
Xrnax.,
Extinctiona,
SIP
hmax.
502
56.8 380 2124
4iO 560
m .If Extinctiona, 560 Alp 58.7 42.9 2124
4 mJf extinction = absorbance c, , where e is expressed as millimoles of amino acid.
solutions treated in a similar manner. Knos gelatin was found to have a very high hydroxyproline content (15.3’%). The hydroxyproline content of the other samples of gelatin and collagen tested ranged from 14.2 to 14.3%, which compares favorably with the literature values included in the same table for comparison. The average coefficient of variation was found to be 1.33 (100 u/m) within samples. This procedure may also be applied to connective tissue determinations in muscle, provided prior estraction is made to reduce the ratio of other amino acids to hydroxyproline. Because tyrosine and tryptophan are known to react like hydroxyproline, the spectral characteristics of the color produced from tyrosine and tryptophan n-ith this method were determined. Five hundred micrograms per milliliter of tyrosine and 100 y per ml. of tryptophan n ere each substituted for hydrosyproline in the analytical procedure described above, and the resulting spectral absorption of the color in the region 425 to 700 mp was recorded. The results given in Table IV indicate 2.00 and 1.30% interference with hydroxyproline a t 560 mp a t equimolar concentrations of tyrosine and tryptophan, respectively. So decrease in absorption maxima of the rolor from t>-rosineand tryptophan n-as observed during 3 hours. ACKNOWLEDGJIENT
Sext, the optimum conditions of heat treatment for maximum color development with 1.55 sulfuric arid were determined. The selection of this acid strength R as based on previous findings although 1.5N and 3 . 0 5 appear equally good (Table I). I n this experiment, absorbance was measured v-ith a Klett colorimeter, using the No. 540 filter. The results given in Figure 1 indicate that optimum heat treatment is 80’ C. for 30 minutes. The procedure given above was applied in a determination of the hydro\yproline content of various gelatin and collagen samples. The results (Table 111)were corrected for the degradation of hydroxyproline n-hich occurred during the acid hydrolysis of the sample, using a calibration curve of standard hydroxyproline
The authors are indebted to Arthur I-eis of Srmour and Co. for a generous supply of cattle hide collagen. LITERATURE CITED (1) Baker, L. C., Lainpitt, L. 11.. Blown, K. P., J . Sci. Food d g r . 4 , 165 (1953). (2) Ibid., 5, 226 (1954). (3) Yeurnan, R. E., Logan, 11. A , , J . B i d . Chem. 184, 299 (1950). (4) Veis, A., Cohen, J., J . Am. Chem. SOC.76, 2476 (19%). (5) Wierbicki, E., Deatherage, F. E., J . AT. Food C‘hem. 2, 578 (1954). RECEIVED for review -4ugust 15, 1955. Accepted January 23, 1556.
Chromatography of the 2,4=Dinitrophenylhydrazones of Some Aldehydes and Ketones in Tobacco Smoke DONALD A. BUYSKE, L. H. OWEN, PELHAM WILDER, JR., and MARCUS E. HOBBS Duke University, Durham, N. C.
A method of general applicahility has been developed for determination of the lower molecular weight aldehydes and ketones in a grossly impure mixture. The procedure was applied to the smoke from bright and burley tobaccos and blends of these, by trapping the smoke at liquid air temperatures and converting the unfractionated raw smoke into 2,4-dini trophenj-lhydrazones. Chromatograms on paper treated with S,,V-dimethylformamide and developed with n-hexane effected separation of the 2,4-dinitrophenylhydrazones of furfural, formaldehyde, acetaldehyde, propionaldehyde, acetone, niethyl ethyl ketone, diethyl ketone, and butyraldehyde. These conipounds were identified by comparative paper chromatography and by their absorption spectra. The free aldehydes and ketones of low molecular weights totaled 3 to 3.3 mg. per cigarette and comprised about l O 7 ~of the total w-eight of the smoke. Acetaldehyde was present in the highest concentration. Only minor variations in content were found in smoke from the types of cigarettes investigated.
T
HE chemical composition of tobacco smoke has long been a
subject, of wide interest and qualitative data concerning the identity of some of the more plentiful compounds are available in the literature ( 3 , 4). This list, numbering over 100 different components, is far from romplete and there still remain a large number of compounds yet to be identified. Much of the avnilable information was obtained under conditions that amounted essentially to a destructive distillation of the tobacco rather than simuhted human smoking. I t is not certain that the information thus obtained is applicable, even in a qualitative way, to tobaccos currently in use. I n the older chemical literature when quantitative data were reported, the authors often failed to define the conditions of smoking, employed nonstandardized methods, or omitted information about the type of tobacco used. Itecently there have appeared quantitative results which were obtained using well-defined standardized conditions ( 7 , 9, 1 4 ) . Because important chemical differences undoubtedly occur with differences in tobaccos and with the conditions under which the tobacco is smoked, much of the older information will be of limited value relative to the quantitative composition of smoke from modern .4merican cigarettes.
