T H E STUDY O F T H E TAX&-IK-GELATIS REACTIOX* BY IMO P. BAUGHMAN
Purpose of Present Work The purpose of this investigation \vas: ( I ) to devise a method for the quantit'ative determination of tannin in small amounts; ( 2 ) to study the tannin-gelatin reaction in t,he vicinity of the maximum flocculation point; and ( 3 ) to determine the effect produced by various electrolytes and by age on this reaction. Part I A Colorimetric Method for Tannin Since tannin gives characteristic color reactions with various reagents under the proper conditions, and since the quantity to be estimated is small, the possibility of a colorimetric method was alluring. Sodium hydroxide was first used but found to be unsuccessful colorimetrically. The characteristic color produced by iron salts with tannin is in many respects ideal, but the difficulty is that many of the salts are so strongly colored that their escess masks to a certain extent the characteristic color sought. The use cf ferrous ammonium sulfate seemed to offer a solution of the difficulty, and an extensive study of the method was made. A sample tannin solution containing o.oooz gin 'cc was diluted still further by adding fifteen cc of distilled water, thus producing a solution containing 0.00012; gm/cc, and then an arbitrary amount of iron reagent was added. One portion was used as a standard, and to the other the iron reagent was added dropwise to test the effect of excess reagent. -1set of readings mas carefully taken after each addition. If the first two readings of each set were not the same, then three to five readings were taken, and the average obtained. The accompanying graph, Fig. I , in which readings in mm are plotted against increasing amounts of iron reagent added to a constant' quantity of tannin, shoivs that a.masimum reading is obtained beyond which an excess of the iron reagent causes no change in color. d solution of tannin containing 0.00033 gni 'cc was diluted to one-half, one-third, and one-fcurth that concentration, and the resulting solutions tested as follows, the Kober t,ype of colorimeter being used throughout this work. Table I gives t,he observed and calculated values in tabular form. In t'he column under the heading "Tannin, gin cc," are given the amounts of tannin present in each sample as determined by dilution from the known solution. In the nest column under Headings are recorded in mm the actual values obtained from the colorimeter for the samples as indicated. In the next column are given the values calculated from the preceding column. Thus *This article is abstracted from the thesis suhmitted to the Department of Chemistry of the Leland Stanford Junior University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.
449
THE TASSIX-GEL.4TIh7 REACTION
in sample number one the more dilute solution was set at a reading of 60.0mm. The other cup should have given a reading of 30.0 mm since the solution it contained was twice as strong as the first. Or assuming that 29.7 niiii was correct then the other reading should have been twice that or 59.4m m . Then from the actual readings taken the amount of tannin present was calculated and recorded in the last column: taking the readings 60.0 and 29. j mm and the o.00033 gni,'cc saniple as standard then, 0.00033 : S : : 60.0: 29.7 s = 0.000163 On the other hand, using the 0.000166giq'cc sample as standard, 0.000166: s : : 29.7 : 60.0 S 0.00033j
FIG. I
TABLE I Actual Tannin __ gm/cc
Calculated Readings ~mm
Readings mm
Tannin gm/cc
3.
0.000330
40.0
39.6
6.
0.000166 0.000330
0.000335 0.000163 0.000334 0.000109 0.000333 0.000082 0.00033I 0.000166 0.00033j 0.000163
19.8
20.0
0.000333
0.000111
60.0
59.4
0.000330
20.
0.000083
80.0
0.000109 0.000330 0.000083
I.
2.
3. 4.
I '
0.000330 o.ooo166
29.;
60.0
30.0 59.4
0.000330
19.9
20.0
0.0001 II
60.0
59.7
0.000330 0.000083
19.9
20.0
80.0
0.000330
30,I
o.ooo166
60.0 19.8
79.6 30.0 60.2
I
20.0
20.0
80.4
1 hus it is seen that the characteristic blue color is produced by the action of ferrous ainnioniuni sulfate on a solution of tannin with an intensity of color which increases proportionally to the Concentration of tannin, provided an excess of the iron reagent is present sufficient to react completely with all the tannin. The readings in mm, when the solutions are compared in the colorimeter, vary inversely as the concentration of tannin in the sample.
450
IMO P. BAUGHMAN
This method has been worked repeatedly for rapidity and accuracy. As a result of much experience with the method the following set of directions has been devised and found satisfactory. The iron reagent is prepared by making a saturated solution of ferrous ammonium sulfate. This solution is filtered and the resulting clear filtrate used as a stock solution. One cc of this reagent is used for a I O cc sample of tannin solution containing not more than o.oooz gm/cc. A concentration of tannin greater than this cannot well be used since it produces too dark a color. Besides the iron reagent it is also necessary to have a standard solution of tannin of known concentration not to exceed o.oooz gm/cc to which the unknown can be compared. The tannin used for solution in these experiment’s was Kalilbaum’s purest product. Ten cc of the standard tannin solution are measured into a test tube; then a measured dolume of the solution containing tannin in unknown concentration is diluted to a total volume of I O cc (a preliminary test having previously been made t o determine approximately the intensity of color) ; I cc of the iron reagent is added to each, and t’he two compared colorimetrically.
Experimental. Part I1 Preparation and Preservation of Dilute Tannin and Gelatin Solutions for Long Periods.-One-half per cent solutions of tannin were prepared in the following manner: 5 gms. of tannin were dissolved in 1000 cc of water, boiled one-half hour, cooled, diluted to 1000cc, poured into tall cylinders, and allowed to st,and until settling was complete. Several hours later the liquid was poured into a 1000 cc glass-stoppered reagent bottle. Any very small particles &ill remaining soon settled and the clear supernatant liquid was drawn off by means of a pipette and diluted as desired. The bottles used for storage must be thoroughly clean and free from foreign matter, otherwise mold is liable to appear within a few days. It is an interesting fact that mold so produced is present in a concentric ring formation. The gelatin used for the solutions in these experiments was the ordinary variety of Knox gelatin, several packages being obtained and the contents thoroughly mixed to assure as uniform material as possible. A sterile 0.5 per cent solution was prepared in the following manner: 5 gms. of gelatin were allowed to stand in zoo cc of distilled water for about 2 0 minutes. Then the mixture was heated until all the particles were dissolved. This was then diluted to I liter and the liquid introduced into a flask of one and a half liter capacity fitted with a two-hole rubber stopper, carrying in one hole a piece of glass tubing penetrating nearly to the bottom of the solution and above the stopper, which was bent like a siphon and closed at the end by a rubber tip and screw clamp; in the other hole was placed a straight calcium chloride t.ube so packed n-ith absorbant cotton that air could just be forced through. The solution was heated in the flask until steam passed through the cotton, the contents being thus sterilized. Portions of the solution were obtained by opening the screw clamp and blowing through the calcium chloride tube.
THE TAXXIN-GELATIiY REACTIOP;
451
This method of preparing and keeping the gelatin solutions was used very successfully, Solutions have been kept over three years and still show no evidence of mold.
The Flocciilntion Point of Tannin and Gelatin Solutions.--Wood' in working with tannin and gelatin found the greater quantity of gelatin-tannin precipitate obtained if about six times as much tannin as gelatin (in two per cent, solution) was present. In 1910TrunkeP reported that mutual flocculation of gelatin-tannin solutions occurs; that a point can be reached at which gelatin and tannin are both completely flocculated; and that a freshly prepared gelatin solution requires more tannin than does a twenty-four hour old gelatin solution. Thomas and Frieden report that gelatin is completely precipit,ated by gallotannic acid when the ratio of tannin to gelatin is not less than z : I , and the gelatin-tannin precipitate is not soluble in excess of tannic acid; and that the ageing of gelatin solutions has no effect on the sensitivity in testing for tannin, provided bacterial action is prevented. For further literature on the Tannin-Gelatin React,ion see Thomas and F r i e d e ~ ~ In order to determine approximately the point of maximum flocculation for tannin and gelatin solutions a set of preliminary tests was made by preparing several series of I O cc samples of the two solutions in varying concentrations and mixing them. Since in many cases the order of addition of colloids makes a difference in their action, any possible complication from this cause was avoided by observing a definite order throughout the experiments. Each set consisted of 9 samples made up in the following way: into the first test tube was introduced I cc of a one-half per cent gelatin solution using a I O cc pipette graduated in tenths; int,o the second, z cc; the third, 3 cc; and each tube in like manner up to and including the ninth. To number one was added 9 cc of a one-half per cent tannin solution; to tube number two, 8 cc of tannin; to number three, 7 cc; each tube in order being treated similarly until at the end there were nine tubes each containing I O cc of a mixture of one-half per cent gelatin and one-half per cent tannin solutions. Each tube was inverted once only to mix thoroughly the contents and then restored t,o its original upright position. Flocculation occurred in each case as soon as the tu-o solutions came into contact. After the precipitates had settled, the first five samples usually showed a clear supernatant liquid; samples six and seven were quite milky and frequently eight and nine were less milky than the preceding two. The samples were allowed to stand a few hours in order that the precipitates might settle to a certain extent, and then each sample was filtered by means of a heavy asbestos pad in a Gooch crucible, The filtrates only were used in all cases, the precipitates being discarded. Ordinarily t'he first half of the series gave clear filtrates, but the following ones were milky in appearance and varying in degree from one series to another. Dekker: "Die Gerbstofie" p. 32 I . Biochem. Z., 26, 458-493 (1910). J. Ind. Eng. Chem., 15, 841 (1923)
452
IMO P. BAUGHMAN
The samples giving milky filtrates were also very difficult to filter. The filtrates were tested qualitatively for tannin and gelatin, the sodium hydroxide and picric acid tests being used respectively. Table I1 gives typical results obtained from testing several series for the presence of tannin and gelatin.
TABLE I1 tannin present I. 2.
3. 4.
5.
6. 7.
8. 9.
>I
11
,)
,f
tf
,,
gelatin absent
,, ,,
,, ,,
12 1,
>I
J,
,, ,,
,J
9)
Jl
,J
,I
,I
1)
,,
,I
I)
,I
,l
,,
I,
J l
,,
doubtful gelatin present
Tannin was thus found to be present in all the samples in each series; gelatin was found present in the latter part of the series from about samples five to nine inclusive.
TABLE I11 Series B
Series A Sample Number
Tannin gm/cc Original 0.00450
Tannin gm/cc Final
Ratio
Tannin gm/cc Final
Ratio
,280
o.oooy9
,298 ,248
0.00126
o.00400 0.003jo
o.00104
,260
0.000;;
,220
o.oooj6
,217
0.00300
0 . 0 0 0 4j
.I50
0.00045
.I50
j.
0.00250
0.00024
,096
0.00027
6. 7. 8.
o.00200
0.00023
.11j
0.00029
O.OOIj0
0.00033
,220
0.0003
O.OOIOO
0.000 I9
. I90
0.0002 I
. 2I O
9.
0.000~0
0.00013
,260
o.00013
,260
I.
0.004jo
0 . 0 0 1 2I
,269
0 . 0 0 123
'274
2.
o.00400
,245
0.00102
.2jj
3. 4.
O.OOjj0
0.00098 0,00081
,232
0.00084
,240
o.00300
0 . oooq7
.156
0,00045
. I50
5. 6. 7.
0.002jo
0.0002;
. IO8
0.00026
0.002oo
0.00023
.IIj
0.0002
,104 ,135
0.0OI~O
0.00020
'
8. 9.
0.00100
O.OOOI2
. I20
1. 2.
3. 4.
0.00134
___
108 I45
,206
Series D
Series C
0.000~0
I
,
I33
__
7
0.00016
,106
0,0001 3
. I30
0 , 0 0 0 1I
,220
THE TANNIN-GELATIN REACTIOS
453
The quantities and concentration were happily chosen, as the apparent point of maximum flocculation is about at the middle of the series. Hence for all subsequent work the same concentration of solutions was used and the quantities made up in like series as indicated above. In order to follow the conduct of tannin-gelatin reactions many series of samples were prepared and the filtrates tested not only qualitatively for tjhe presence of tannin and gelatin but also quantitatively for the amount of tannin using the colorimetric method devised. Quantitative Study of the Tannin-Gelatin Reaction.-The colorimetric method described in Part I for the determination of tannin in small amounts was now used in studying the tannin-gelatin reaction. The method here \vas identical with that used in the preliminary qualitative work, except that the tannin in the filtrates was determined quantitatively by means of the colorimetric method described in Part I. Table I11 shows the analysis of the filtrates from four series of samples. The sample number is the same as the number of cc of gelatin in that particular sample and the total volume of each sample is I O cc. In the “Ratio” column are given the ratios of tannin found in the filtrate to the tannin originally present in the sample. From a study of these results it is seen that when tannin and gelatin are mutually flocculated, there is a point of maximum flocculation beyond which with increasing gelatin some flocculation st’ill occurs, but a decided protective action also comes int,o play. The largest amount, of tannin found in t,he filtrat,e is from z j to 30 per cent of the original quantity in sample number one. The quantity decreases rapidly in the following samples to number five at which point the smallest amount of tannin is found-about I O per cent of the original quantity. In samples six to nine inclusive the tannin content of the filt,rates increases though not quite so rapidly as it decreased at the beginning of the series, about 2 5 per cent of the original amount remaining in the ninth sample. The Efect of Electrolytes on Tannin-Gelatin Reactions.-The effect on tannin-gelatin reactions of electrolytes in varying concentrations was observed in the following way: a 6 per cent solution of the elect,rolyte was prepared, and z cc of this solution added to every t,est tube in the series of 18 samples, making in each tube a total volume of I Z cc of tannin and gelatin in varying proportions but always with a I per cent solution of the electrolyte present. Later a 1 2 per cent solution of the electrolyte was prepared, which when added to the two sets of a second series of I O cc samples in portions of z cc gare a salt concentration of 2 per cent; and finally a 24 per cent solution of the electrolyte was used giving the effect of 4 per cent salt present in the dilution used. The electrolytes were chosen particularly with respect to the anions and cations t o see if there was any noticeable difference in their action. Certain salts could not’ be used on account of interference in various ways with the necessary colorimet,rictests. Barium salts had t o be eliminated on account of the precipitate fbrmed with ferrous ammonium sulfate and colored salts also could not be used on account of interference with the characteristic color of the iron tannate to be formed. The results of duplicate sets are given in Tables IV-XIII.
454
IMO P. BAUGHMAN
I
Sample Number
TABLE IV per cent sodium chloride Series A
Tannin gm/cc Original
Tannin gm/cc Final
jo
I.
0.004jo
0.00123
.2
2.
o.00400
0.001I O
' 2 i 5
3. 4.
0.00350
O.OOOj0
,144
o.00300
0.00032
j.
0.00250
0.00017
6. 7. 8.
0.002oo
9.
Tannin gm/cc Final
Ratio
0.00112
,249
0.00094 0.00044
,126
,107
0.00027
,090
,068
0.0001~
,060
0.0002 7 o . 0001 8
"35
o.00030
,150
. I20
0.00024
,160
O.OOIOO
0.00014
,140
0.0001j
. I jo
0.000j0
0.00012
,240
0.00014
,280
O.OOIj0
2
Sample Number
Series B
Ratio
TABLE V per cent sodium chloride Series h
Tannin gm/cc Original
Tannin gm/cc Final
'235
Series B
Ratio
Tannin gm/cc Final
Ratio
I.
0.004jo
0.00133
,296
0.00127
,282
2.
o.00400
0.00133
0 .OOI I 8
,295
3. 4.
0.003jo
0,00092
,263
o.00300
0.00098 0.00069
'332 ,280 ,230
0.00044
,146
5.
0.00250
0 . COO33
I32
0.0003I
. I24
6. 7. 8. 9.
0.00200
0
00043
. 2 1j
0.0004 j
. 2 2j
O.OOIj0
0.0003 2
,213
0.00032
,213
O.OOIOO
0.00024
,240
0.000~0
0.00019
380
4 Sample
Tiumber I. 2.
3. 4. 5.
6. 7. 8. 9.
Tannin w/cc Original 0.004jo
T.4BLE VI per cent sodium chloride Series h
Tannin gm/cc Final
Ratio
0.0013;
.30c ,308 2 86
o,00400 0.003 jo
0 . O O I 23
0.00300 0 . 0 0 2j o
0.o o o j o 0.00035
0.00200
0.00048
0 , O O I00
'
233
,140 ,240
o
,230
00023
,380
0 . O O O I9
Series B Tannin gdcc Final
Ratio
0,001 44
,320
0.00123
,308
0.00099 0.00046 0.00034
. I j3
0.00028
,140
,240
,283 ,136
0 . 0 0 150
0.00033
,220
0.00036
0.00100
0.00022
,220
0.00024
,240
0 , 0 0 0j
O . O O O I6
,320
0.00020
,400
o
THE T A S S I S - G E L A T I S REACTIOS
455
TABLE VI1 I
Sample Sumber
Tannin gm/cc Original
per cent sodium sulfate Series A Tannin gmi'cc Final
Ratio
I.
o.oojj0
0,OOI92
2.
0.00~00
0.00142
,427 .3jj
3.
0.003jo o.00300
0 .OOI
08
,308
4.
:.
0.002jo
6'.
0.00200
0.00063 0.00034
,210
,136 ,190
;.
O.OOIj0
0.00038 0.00033
$8.
0.00I00
0 . 0 0 0 2j
,250
SI.
0.000j0
O.OOOI4
,280
I
Sample Sumber L .
Tannin gmlcc 0rigin a I o.ooqj0
2.
0.00~00
J.
'+.
0.003jo o.00300
,j.
0.002jO
15.
0.00200
7. 13. 9.
0.001j0 0.00100 0.000~0
TABLE VI11 per cent magnesium chloride Series d Tannin gmlcc Final o.00161 0.00161
Ratio
0 . O O I03 0.00068 0.00037 0.00061
,293 ,246 ,148
0.00044 0.0003z 0.0001 j TaBLE 2
Sample Sumber
,220
,358 .40I
Tannin gmlcc Final
Ratio
,360 ,330
oc103
,294
O.OOOj8
,193
,144 . 3 I5
,293
0.00036 0.00063 0.00038
,320
0.00030
,300
,300
0.00018
,360
'
305
0.
.2j3
1s
per cent magnesium chIoride Series A
Tannin gm/cc Original
Series B Tannin d c c Final 0.00162 0.00132
Series B
Ratio
Tannin gm/cc f1n31
0.00139 0 . O O I 16 0.0009j
Ratio
I.
0.004jo
0.00095
,211
2.
o.00400
0 , O O I2 0
,300
3.
0.00350 o.00300
0.00094
4.
0.00047
,268 ,156
0.00043
5.
0.00250
0.00025
,100
0,00024
,096
6. 7.
0.00200
0.000 jI
.2jj
0,00045
,225
O.OOI50
0.00036
,240
o.00030
,200
0.00I00
0. on024
. 2.40
0.00024
,240
0.000~0
0.00020
,400
0.00017
,340
8. 9.
,309 ,290 ,272
143
TABLE
1
4 per cent inagncsium chloride Series A Sample Kurnber
Tannin gmlcc Original
Tannin
Ratio
Series B Tannin
Ratio
gm/cc
gm/cc
Final
Final
I.
0,004iO
0 . 0 0 150
,333
0.00I 2 j
,282
2.
0.00~00
,290
0.001 II
,277
3.
0 . 0 0 3j o
0.00166 o 00096
,274
0.00088
.2ji
4.
0.00~00
0.00043
1-13
0.00O~I
3.
0.
,092
0.00024
,136 096
6.
0,00200
0.000~2
,210
0.00042
,210
I '
0.
oo1:o
0,0003 I
,206
0.0003;
8.
0.00100
0 . 0002 ,j
,250
0.00024
9.
0.000j0
0.00016
,320
o 00018
233 240 ,360
ooz50
0.0002~
I
Sample Number
TABLE SI per cent nluniinuin chloride Series -1
Tannin gm/cc Original
Tannin
Ratio
gm,'cc
Final
Series R Tannin gm/cc Final
Ratio
I.
o.oo~~jo
o
00129
,266
0 , 0 0 119
,264
2.
0.00400 0.003.;0
0.0010;
26;
o.OoI0;
,262
0.000.: j
13;
0 . oooj4
0.00300
0.00046
. I ,i3
0.00039
,
5.
0.002.
