A. A. B E N E D E T T I-P IC H L E K 1 AND M IC H A E L C E F O L A l W a sh in g to n S q u a re College, New Y ork U n iv ersity , New Y o rk , N . Y.
T h e te c h n iq u e o f w o rk in g in th e c a p illa ry cone h a s b e e n e x te n d e d to p e r m it t h e p e rfo rm a n c e o f c o n firm a to ry te s ts a f t e r t h e se p a r a tio n o f c o n s titu e n ts . T h e te s ts a r e c a rr ie d o u t o n a c ir c u la r p la te a u o f 0.03- to 0.2-s q . m m . a re a o b ta in e d by c u t tin g a g la s s th r e a d o f 0.2- to 0 .5 -m m . d ia m e te r . T h e th r e a d is d ra w n o u t fro m th e e n d o f a s h o r t glass ro d o f 5- m m . d ia m e te r , w h ic h serves a t th e sa m e tim e a s c o n d e n s e r fo r c o n c e n tra tio n o f th e lig h t in th e p la t e a u . T h e id e n tific a tio n o f a p p ro x im a te ly 0.001 m ic ro g ra m o f th e m o re c o m m o n io n s o f th e h y d ro g e n su lfid e g ro u p is a s s u re d in th is m a n n e r .
T h e cell fo r th e p e rf o rm a n c e o f th e o p e ra tio n s h a s b een m odified to a c c o m m o d a te a g re a te r \ a r ic t ) o f tools, a n d b e t t e r c o n tr o l o f th e o p e ra tio n o f th e m ic ro p ip e t h a s b e e n o b ta in e d w ith th e u se o f a p lu n g e r a d v a n c e d b y m e a n s o f a screw w ith fine th r e a d .
A
P R E C E D IN G p a p e r (2) described a general working technique, w hich can be applied to th e q u alitativ e analysis of sam ples of 1 m icrogram m ass or less. T he procedure em ployed in sep aratin g th e c o n stitu en t ions was ou - lined, b u t th e perform ance of confirm atory tests w as n o t men
tioned. T h is re p o rt m ay be considered an extension of ■ e first paper, dealing w ith confirm atory te s ts a n d some raodm - cations of th e w orking technique. V a r i o u s changes in th e e- sign of a p p a ra tu s are in p a r t required to facilitate the per form ance of confirm atory te s ts an d in p a r t suggested by ie accum ulation of experience in work of th is kind.
A p p aratu s
C a r r i e r . To enable th e mounting of a larger number of re agent capillaries, capillar,' cones, etc., the dimensions u . rier slide have been changed to 35 X 25 X ‘ J ;mPnii0n3 cut from plate glass, or microscope slides of the proper d _. - may be cemented together to give the thickness desir . thick slide is easier to handle and has the further a d v a n ta ^ th a t it supports the capillaries in approximately th e level l
platform of the condenser rod is located.
M o i s t a n d D r y C h a m b e r . The all-glass chainber rccom mended for biological work by Chambers (5) and used in the pre
p r e s e n t address, Queena College, Flushing, N . ' i .
1 Present address, M etallurgical L ab o rato ry , T mvers» > o Chicago, 111,
liminary experiments (2), was found unsatisfactory for two rea
sons- (1) I t does not provide ample room to accommodate a condenser rod and a capillary carrier of the above-specified di
mensions which would comfortably hold as m any as six or seven capillaries without crowding; (2) it causes difficulties in manipula
tions, especially in removing and introducing capillaries.
The new chamber shown in Figure 1 has been found entirely satisfactory. The sides, a, consist of pieces of brass 67 mm. in length 6 mm. in thickness, and 11 mm. in height. Four grooves run lengthwise, and, in addition, each bar has a vertical groove, f at one end. T he two pieces forming the side of th e chamber are attached with screws to a brass frame, b, which in tu rn is m ounted by means of De K hotinsky cement on a glass plate ft (68 mm. X 60 mm ) serving as the bottom of the chamber. The brass frame is 1 mm. thick and made to fit plate k, so as to give a border 5 mm wide all around. This leaves an area approximately 48 X o7 mm through which light can pass. A piece of glass slide, c, which fits the vertical grooves, f , forms the back of the cell. I he top elate d is cut from an old projection slide, so th a t it can be freely moved when inserted into the upperm ost of the horizontal grooves of the brass bars.
Thus, the dry chamber resembles a chess box with the cover sliding in and out and one end open. To convert it into a moist chamber, the remaining horizontal grooves of the brass bars are lined with cotton, e, which, if slightly moistened w ith w ater, produces a dam p atm osphere in th e cell.
Fo r c e p s. At least two forceps, one of which should be cork-
tiDDed are required. T hey should be made of stainless steel and have smooth (not corrugated or toothed) parallel tips.
Cork-8 1 3
tipped forceps are necessary, for they prevent the loss of capil
laries which often slip th e grasp of plain forceps. T he loss of a capillary cone m ay m ean the untim ely end of an investigation on which a g reat deal of work has already been spent.
Il l u m in a t i o n o f t h e Mi c r o s c o p ic Fi e l d. Quick change from observation w ith transm itted light to observation w ith re
flected light is most conveniently obtained by m eans of two m i
croscope lamps. Any inexpensive source of light m ay be used for the illum ination of the m irror of th e microscope. T he second lam p should be equipped w ith condenser lens and diaphragm , so as to give a narrow pencil of intense light which is sent in a hori
zontal direction through the rear window (c, Figure 1) into the interior of th e chamber. To this end, the lam p is placed on one side of the microscope and m ounted a t the height of the stage of the microscope. Switches for bo th lam ps should be located side by side w ithin easy reach of the experimenter.
Mi c r o p i p e t s. M icropipets having a bore of approxim ately 1m a t the opening were used a t th e outset and operated by means of a hypodermic syringe according to the directions of Chambers (5). i t was found more convenient in work w ith capillary cones to give th e micropipet a wider opening and to control th e flow' of liquid mechanically by m eans of a plunger, the motion of which is regulated by a screw w ith fine thread. T he micropipets were prepared w ith the use of liachele’s device U ) , and a portion of the gradually tapering fine tip was snipped off w ith a pair of forceps to give an opening 3 0 to 4 0min diam eter.
Pl u n g e r De v i c e. F o r use w ith m icropipets of th e above in
dicated wide opening a t the tip, the plunger device designed by Johnson and Shrew sbury (6) was adopted. I t is visible in the foreground of Figure 2. A flexible copper tubing connects the tu b u lar cham ber w ith the pipet holder in th e clamp of the m anipu
lator. T he plunger is operated by rotation of the milled head.
T ubular chamber, copper tubing, and pipet holder are filled with distilled w ater from which air has been removed by boiling. The w ater is allowed to occupy the first third of th e shank of the mi
cropipet. T he rest of the shank remains filled w ith air which serves to separate the liquids in th e m icropipet proper from the hydraulic w ater.
Re s e r v o i r f o r Cl e a n i n g Fl u i d. A short b u ret is m ost con
venient for use in cleaning micropipets. A fresh drop of cleaning fluid, usually w ater, is supplied for every rinse, so th a t it remains
814 I N D U S T R I A L A N D E N G
F i g u r e 2. W o r k i n g i n t h e C a p i l l a r y Co n e
hanging from th e tip of the buret. T he micropipet is introduced into th e hanging drop, and suction is applied w'ith the plunger.
When enough liquid has been taken in to fill shaftlet and taper, the pipet is w ithdraw n from th e drop of cleaning liquid ana its contents are expelled b y touching a piece of filter paper to the opening of th e micropipet and reversing the m otion of the plunger.
1 he manipulator used perm its rotation around its vertical column as axis. T he b u re t w ith th e cleaning fluid is placed so th at it is only necessary to w ithdraw th e m icropipet from th e moist cham
ber and ro tate the m anipulator through 90 degrees to get the pipet to th e tip of th e buret.
N E E R I N G C H E M I S T R Y Vol. 14, No. 10
Fi g u r e 3. Co n d e n s e r Ro d
Co n d e n s e r Ro d. T he condenser rod used in the performance of confirmatory tests is made of glass which shows only a little color when viewing through a layer 3 cm. thick. A rod about 4 to 5 mm. in diam eter is draw n out to a finer rod (6, Figure 3), which is then cut so as to leave approximately 7 cm.
attached to th e original rod. N ext, b is heated a t a distance of 10 mm. from the tap er and pulled out to a fine thread, c, of 0.2- to 0.5-mm. diam eter, which is cut 20 mm. from the taper. The bend a t c is obtained by holding th e rod horizontal and approach
ing th e thread w ith a sm all flame. If not too much heat is ap
plied, th e end of th e thread will slowly swing through 90 degrees as it follows the pull of gravity. T he original rod, a, is next cut to a length of 10 to 12 mm., scratched w ith a file, and then broken while held a t a by m eans of a pair of pliers. Thread c ¡9 then dipped into m olten paraffin and slowly w ithdrawn again.
When the paraffin coating has solidified, th e thread is cu t approxi
m ately 5 mm. above the oend and broken ofT w ith forceps. The break should be clean and, as closely as possible, a t a right angle to th e axis of th e thread. B y m eans of some D uco cement, the rod is finally m ounted on a sm all cover slide, «, so th a t the thread is vertical to the cover slip.
C o n firm a to ry T e sts
Q u a lita tiv e an aly sis of sam ples of 1-m icrogram m ass must p e rm it th e identification of 0.001 to 0.01 m icrogram of a minor c o n stitu e n t. T hese q u a n titie s a re sm aller th a n th e lim its of id en tificatio n of m o st slide te s ts, a n d i t follows t h a t general use of th e c u sto m ary slide te s t tech n iq u e is im possible with sam ples of 1 m icrogram m ass. T h e lim it of identification of th e slide te s ts can be im proved to m eet th e requirem ents by reducing th e volum es of th e te s t d rops to 0.01 to 0.001 cu.
m m . E v en th en , th e cry stals of th e p re c ip ita te s a re so small, for lack of m aterial, t h a t as a ru le th e ir sh ap e can no longer be safely discerned u n d e r th e m icroscope. T h u s, th e use of th e cry sta l form a s criterion for th e identification frequently h as to be ab an d o n ed , a n d one is th e n reduced to th e criteria em ployed w ith te s t-tu b e te s ts : ap p earan ce or disappearance of phases, a n d color phenom ena. A s in te s t-tu b e experim ents, preference will be given to te s ts w hich give characteristically
and intensely colored p recip itates an d th u s assure a high de
The condenser rod (Figure 3) is placed in the moist chamber, so that the circular end surface of rod a faces the rear window of the chamber and is close to it. The condenser rod as well as the car
rier, which is placed next to it, is k ep t from sliding about by wet
ting the under sides w ith a small drop of water. The film of water forming between th e base plate of the chamber and the glass plates of the apparatus holds the latter in place. The moist chamber is mounted on th e stage of the microscope so th a t the rear window faces the lam p mounted on the side of the stage, and the pencil of light is concentrated on the end surface of rod a.
Once in the rod, m ost of th e light is held there because of total reflection, concentrated into th e narrow thread, and led around the bend so th a t it emerges as a narrow, vertical bundle through the small end surface, d (7). The tests arc carried out on this end surface which has been called the “ plateau” . The paraffin coat
ing of thread c prevents th e te s t drops from flowing over the edge of the circular surface, and in this way it is possible to confine the drops w ithin areas of 0.03 to 0.2 sq. mm., depending upon the diameter of c. In m ost of th e work reported below7, plateaus of limited to approxim ately 20, and the plateau will not fill the field of vision of th e microscope.
Test solution and reagent are transferred to the plateau by means of the micropipet. The plateau is focused with the micro
scope, and then the m icropipet is guided with the manipulator, so th a t its tip appears in sharp focus close to the plateau. The pipet is slightly raised and moved horizontally, so th a t the open
ing of the tip appears above the plateau about half way between the center and the near edge of th e platform (Figure 4, left).
The pipet is next lowered so th a t it actually touches the platform and the image of the tip again becomes sharply defined. Applica
tion of pressure by means of th e advancing plunger delivers the solution from the m icropipet to the plateau. The pipet is then raised and withdrawn. T he sam e general procedure is followed in adding reagent solutions. Slight pressure, however, is applied with the plunger before the opening of the micropipet is lowered into the te st drop on the platform. This sta rts th e outflow of the reagent the m om ent the opening of the pipet makes contact with the test solution, and clogging of the pipet by formation of pre
cipitates inside the tip is prevented.
T he use of solid reag en ts as in slide te s ts is n o t practical.
I t is obvious t h a t tin y particles of som ew hat hygroscopic substances will liquefy im m ediately w hen introduced into the m oist cham ber. M ore im p o rta n t are th e difficulties connected solution of potassium dichromate. T he characteristic reddish- brown precipitate separated immediately, and a num ber of spear- shaped crystals could be discerned (Figure 4, right).
M e r c d r y . Mercuric chloride solution containing 0.2 mole of nitric acid per liter was transferred to the platform of the con
denser rod and treated w ith a slight excess of a freshly prepared solution of 1 p art of s-diphenylcarbazide in 100 parts of ethyl al
cohol. The violet precipitate was observed with a magnification of 150 diameters. The alcoholic solution of th e carbazide assumes a light pink coloration on standing. I t then becomes rath er dif
ficult to differentiate between the color of th e reagent and th a t of the precipitate.
L e a d . Triple nitrite reagent was prepared following the di
rections of Adams, Benedetti-Pichler, and B ryant (I). To prevent dilution, the container with this reagent was introduced into the moist chamber ju st previous to use. Four volumes of a solution containing approximately identical quantities of lead and copper were treated with 1 volume of the reagent solution. T he charac
teristic black squares and rectangles of K jP bC u(N 03)j separated in a short time. W ith 0.001 microgram of lead it was necessary to cause partial evaporation of the final te s t drop by opening the top of the chamber somewhat.
B i s m u t h . A black precipitate of metallic bism uth was obtained immediately on adding a small excess of freshly prepared stannite solution (3) to solutions of bismuth nitrate.
Alkaloid iodobismuthites were investigated in the hope of get
ting strongly colored precipitates, and i t was discovered th a t quinine permits immediate differentiation between bism uth and antimony because of the difference in the color of the precipitates.
One gram of quinine bisulfate is dissolved in 50 ml. of warm w ater to which a drop of concentrated nitric acid has been added. A second solution is prepared by dissolving 2 grams of potassium iodide in 50 ml. of water. Small quantities of tne reagent are made just previous to use by mixing equal volumes of the two solutions.
Discoloration and separation of a precipitate will occur, if the ready mixed reagent is stored for more th an a fewT days. In the preformance of the test, the bism uth solution is treated w ith an equal volume of the reagent. A brown precipitate of quinine iodo- bismuthite separates immediately, and some characteristic spherulites may usually be recognized. R ight. P recipitate of silver dichrom ate
C o p p e r . The copper solution was made to evaporate on the plateau of the condenser rod by p artly opening th e top of the moist chamber. The residue was then treated with a volume of a solution of salicylaldoxime (1 gram in 5 ml. of ethyl alcohol and 95 ml. of water) equal to the volume of te s t solution used. A reenish-yellow precipitate was easily discerned w ith low' magni- cations.
Cupric mercuric thiocyanate was precipitated by treating the copper te st solution with an equal volume of ammonium mercuric thiocyanate reagent (8 grams of mercuric chloride and 9 grams of ammonium thiocyanate dissolved in 100 ml. of w ater). The
yellow precipitate obtained w ith 0.001 microgram of copper was distinctly visible when using a magnification of 150 diam eters.
C a d m iu m . The te s t solution was treated w ith an equal volume of a solution of sodium sulfide (10 grams of sodium sulfide nova- h ydrate in 100 ml. of w ater). The yellow sulfide precipitate ob
tained w ith 0.01 microgram of cadmium was distinctly visible w ith th e use of low magnifications.
A r s e n i c . The te s t solution containing arsenate or arsenic acid was evaporated on the plateau of the condenser rod by partly opening the top of the moist chamber. T he residue was treated w ith 0.001 cu. mm. of silver n itrate in acetate buffer solution (1 gram of silver n itrate, 7.7 gram s of amm onium acetate, and 6 ml.
of glacial acctic acid in 200 ml. of w ater). The reddish-brown precipitate of silver arsenate was easily discerned under low mag
nification.
A n t i m o n y . T he acid te s t solution was treated w ith the qui- nine-potassium iodide reagent as outlined for bism uth. A yellow precipitate was obtained.
Tin. T he solution of stannic chloride was treated on the plat
form of the condenser rod w ith an equal volume of concentrated hydrochloric acid and then w ith a like volume of a satu rate d solution of rubidium chloride. After adding the reagents, the top of the cell was slightly opened to cause partial evaporation of the te s t drop. C rystals of rubidium chlorostannate separated in a
816 Vol. 14, No. 10
sh o rt time. Using a magnification of 150 diam eters, there was difficulty in discerning the shape of the crystals when Ö.001 micro
gram of tin was taken for th e test.
L ite r a tu r e C ited
(1) Adams, J. I., Benedetti-Pichler, A. A., and B ryant, J. T „ M ikro
chemie, 26, 29 (1939).
(2) Benedetti-Pichler, A . A ., In d. En g. Ch e m., An a l. Ed., 9, 483 (1937).
(3) B enedetti-Pichler, A. A., Z. anal. Chem., 70, 257 (1927).
(4) B enedetti-Pichler, A. A., and Rachele, J. R., I n d . E n g . Chem., A n a l . E d ., 12, 233 (1910).
(5) Chambers, R ., in M cClunß's “ Handbook of Microscopical Tech
nique” , New Y ork, P. B. Hoeber, 1929.
(0) Johnson, D. L., and Shrewsbury, C. L., Mikrochemie, 26, 143 (1939).
(7) Wood, R . fW., “ Physical Optics” , p. 69, New York, Macmillan Co., 1936.
Pr e s e n t e d in p a rt before th e D ivision of M icrochcm istry a t the 100th M eeting of th e Am e r i c a n Ch e m i c a l So c i e t y, D e tro it, M ich. Abstracted from p a rt of th e thesis su b m itte d by M ichael Cefola to th e faculty of the G ra d u a te School of N ew Y ork U niversity in p a rtia l fulfillm ent of the re
q u irem en ts for th e degree of doctor of philosophy.
I N D U S T R I A L A N D E N G I N E E R I N G C H E M I S T R Y