Automatic Recording of Spectrophotometric Titration Curves with the Beckmain Model DU Spectrophotometer A.
L.
Underwood and Thomas
M. Robertson, Department of
rr
~ m m :BASIC APPROACHES to the pc~formancr of photometric titration.; :we the manual plotting of a series of individual measurement's, aut,omatic t i t r x i t shutoff a t the m d point utilizing tlie amplified (and sometimes differc i i t i a t d ) signal from the photodet,ector, and automat'ic recording of the bitration c u r n ~ . The last' technique possesses t,lie :draiitage.- of spee j and convenience on'r thr, first. anti it is Iwtter suited than tile second to 1 c s hvorable titration reactions bordering upon nonfeasibility \ h e r e photomet,ric titrations are best :ihk t'o demonstrate tfieir inherent s u p riority to other end po:.nt methods. liitoniatically rwordcd photometric titration curves h u v ~ been reporbed, hnsed upon a Cary iecording spcctrophotomrter (2q3 ) . a 1:erkman Node1 I3 e!,ectroi,liototiiet(~r (4,and instrument:it'ioii iticorporatirig ksoth the Beckman 11odel H and t,he Precision-Dow Kecordoniat,io titrator (1, 5 ) . An autotixitic rwording photometric titrator 1 7 s now been assemb1l.d based upon the Beckman illode1 DU sl)ec:trophotometer, 3 log rrcorder, and a constant rate hurct. Aill of the components can be purc1i:tsed commercially, and no knowledgc of electronics or -.nstrument design is rcquired. Rapid and convenient titr:itions can be p:rformed over a \ d e r n-avelength range and with higher prcciaion than was possible with any h i t t h r most expensive setups previously dcwibet-l. Examples of acid-base, reclor. :inti c~helomctric titrations, both
Chemistry, Emory University, Atlanta 22, Ga.
with and nithout indicators, arc presented which demonstrate the capability of the in.trument.
i
60 4 r
APPARATUS
A Sargent constant rate. motor driven buret (KO. S-11120-1) was used for titrant delivery; this buret has a 10-ml. capacity and delivers a t the rate of 1 nil. per minute. Titrant \\as introduced into the titration v e w l tlirougli Tygoii tubing terminating in a glasq capillary tip. The tubing n a s wrapped n i t h black tape as a precaution againbt stray light. The Model DU cell compartment was replaced by a larger one described previously (6) n hich accommodated a 150-ml. beaker of iiltral-iolettransmitting Corning T'ycor 7910 glass; the path length through the beaker was 5.5 em. Magnetic stirring was eniployed as before (6). This cell conipartment is mounted by nieans of thc. four screws that are ordinarily on hand to attach the phototube housing and large cell compartments to tlie plate on the 1Iodel DU monochroniator case. Thuq it takes only a niiiiute or two t o return the inqtrumeiit t o it. normal condition, and it remains readily available for other measurement>. The Beckman Energy Recording Adapter (ERA, S o . 5800) n a s n-ired into the DI' according to the instructions provided. This is an easy 017eration requiring less than half a n hour. The ERA\ has a simple circuit. and an inexpensiT e vbstitiite coiild be built very ea\ily. A w i t c h on the ER-4 restores: the DU circuit to it-
Table I.
I
1
2
3
I
Milliliters of Titrant
Figure 2.
Titration curves
Lower curve. 104 mg. Fe+a titrated with 0.1N K2CrZ07 at 440 m p ; no indicator titrated with 0.1M Upper curve. 15 mg. C U + ~ EDTA ot 745 rnp; no indicator
iiornial operation. Two different log recorders ir'ere used in the present o-ork: good results were obtained Jr'ith both the Sargent Model SRL and the Photorolt Linear/Log Varicord 43. If a log recorder were not available, a simple, casily constructed, and inexpensive logarithmic attenuator circuit such as that described by Marple and Hume (4) could presumably be used with the other instrumentation described here. RESULTS
Figures 1 and 2 show typical titration w r r e s obtained with the instrument cleacrihed above, and Table I sumniarizes the results of a large number of acid-base, redox, and chelometric titration-. The precision is seen t o be
Results of Titrations So.
NaOH Sa,C03 Sa2C0. As
[&ant it y, mg. 30-2.50 4-3.5 33-93 33-93 30-150 as
Fe -: I:e
50-350 .50-350
CU --
16-33 15-35
Substance titrated KHP
-
I
+A
-
-
vu--?
i
3 Milliliters of NcOH Solutlon
Figure 1 . Titration of potassium acid phthalate with sodium hydroxide 83 mg. KHP titrated with 0.1 N N a O H ot 550 m p
?;1
1-i +?
Ca+2
Iridictztor l'henulplithalein Phenolplit halein Plien olplit halein Methyl red
Tnne
. . i 0i
550 650 640 320
9 12 10 10 8
3 ,4 4.9 6.0 6.4 3.4
AS203
I-
L
Titrant
TVave- of Av. length, titra- dev., tions p.p.t. rnp
+ ?\Ig+Za
Sone
lliphen,~-lnmine
410 .5 85
sulfrinate Sone
T43
8
440
8
7.6 4.8
1000 440
14 14
6.6 4.7
640
6
7 0
Pyrocatechol violet
60-220 60-220
Pyrocatec,hol
0.6-4.0 as
Eriochrome black T
Sone
violet
CaCO3 Water-hardness titratiun using local tap r a t e r .
with phenolphthalein indicator
VOL. 35, NO. 1 1 , OCTOBER 1963
1761
somewhat less than that of the best visual titrations, but it is considered good for a n automatic titrator. The principal limitation is that the titration reaction must be rapid, a condition which must be satisfied in any automatically recorded titration. Because the instrument is assembled largely from commercial components, the worker Iyho is unsophisticated in instrumentation or is not even primarily a chemist but who needs a titrator to
serve his own ends may readily make use of it. If a Model DU spectrophotometer is a t hand, the cost is modest by present standards; if both a DU and a recorder are available, the cost is small.
(3) Malmstadt, H. V., Roberta, C. B., Zbid., 27, 741 (1955). ( 4 ) Marple, T. L., Hume, D. K.,Ibzd., 28, 1116 (1956). ( 5 ) Mullen, P. \Y.,i2nton, A , , Ibzil 32, 103 (1960). (6) Underwood, A. L., Hove, L II.,111, Ibid., 34, 692 (1962).
LITERATURE CITED
WORKsupported by the Sntional xieIice Foundation through Research G r s n t SSF-G13514. Taken from a thesis presented by Thomas XI. Robertsun in partial fulfillment of the requirements for the 11S degree, Emory Cniversitp. 1962.
(1) -4nton, -4.1 hfulleni p. w.2 Talanta 8, 817 (1961). ( 2 ) Malmstadt, H. E. -kXAL. C H E h I . 26, 442 (19%).
v.,
c.,
~
Sample Injector for Ion Exchange Chromatography and Flow Cell for Continuous Photometry at 210 Millimicrons Arthur M. Crestfield, The Rockefeller Institute, New York 2 1, N. Y.
often desirable to determine the I concentration of substances in the T IS
effluent from a fractionation column by measurement of absorbance in the ultraviolet. While automatic sampling photometers are available for use with fraction collectors, it is simpler to pass the effluent through a flow cell in a recording photometer. However, optimal separations are obtained only if the flowing stream is of sufficiently narrow cross-sectional area. The construction of a micro flow cell for use with a Zeiss PMQ I1 spectrophotometer and columns of about 1 cm. or larger in diameter is described here. This equipment has been used to advantage in studies on ribonuclease reported elsewhere (2, 3). I n that work, the sensitivity in detection of proteins was increased b y use of 210 mp (4) rather than 280 inp as usually employed. To obtain a steady baseline, it was necessary to keep the flow rate constant a t all times, even during addition of the sample, since the ion exchange resin itself released materials mhich absorb a t 210 mp. The device developed for injection of the sample onto the column provides other advantages to make it of more general interest. Therefore, this sample injector is described here too. EXPERIMENTAL
Chromatographic Unit. I n t h e prese n t work, a solution of S a C l is emSpring clamp
Teflon c o i l
Figure 1.
1762
Sample injector
ANALYTICAL CHEMISTRY
ployed as eluent. Only the stationary phase of the column (a carboxylic resin) acts as the buffer. The procedure for the preparation of such a column is described elsewhere (3) ; other solutions may be used provided the absorbance at 210 mp is sufficiently low. Columns with a n internal diameter of about 0.9 cm. were equipped with a supporting disk of porous Teflon (Grade 50-55, Fluoro-Plastics, Inc., Filbert and Cuthbert Sts., West of 36th, Philadelphia 1, Pa.). Disks of porous Teflon are added t o 50% aqueous acetic acid, deaerated with a water aspirator for 30 minutes, rinsed several times with a solution of SaC1, and then deaerated again, this time without acetic acid. The bottom of the column is a 12/1 semiball joint. -1 PVC connector of the type used on the Spinco amino acid analyzer (Beckman/Spinco swivel fitting 120-558, PS'C 120-561 connector, 12-mm. socket to 1 / 1 6 inch 0.d. Teflon tubing) provides convenient attachment of the 22-gauge Teflon tubing (Pennsylvania Fluorocarbon Co. Inc., 1115 N. 38th St., Philadelphia 4, Pa.) leading to the flow cell. The detection system is sensitive to air bubbles which appear in the effluent stream %-hen air pressure is used to drive the sample into the column at the beginning of a chromatogram. Some other means of adding the sample is needed, therefore. The photometer also detects any alteration in the small, steady concentration of ultraviolet absorbing material leeched from the resin and the other components. The concentration of the extraneous ultraviolet absorbing material in the effluent is dependent upon the rate of flow through the system. During operation, the flow rate, therefore, must be held constant by a suitable pump. A MiltonRoy minipump fitted with Teflon or polyethylene tubing, and a pressure gauge (200 p s i . , E. S. Gauge) is operated a t 30 ml. per hour (6). A section of silicone tubing of l / l a inch i.d. and length of 6 inches is inserted in the delivery line to provide a n elastic component to reduce the magnitude of
Figure 2. Detail of T-connections shown in Figure 1
the pulse of pressure during the stroke of the piston. A section of Tygon tubing is slipped over this silicone tube to prevent it from bursting. Sediment and cations emerging from the pump are removed by a 6-cm. column of coarser IRC-50, equilibrated in the same manner used for the chromatographic column. The effluent from this column of coarser resin is directed to the chromatographic column. The sample is inserted into the pumped stream nhich feeds the chromatographic column by the use of the arrangement shown in Figure 1. The sample is taken up in a coil of about 10 feet of 22-gauge Teflon tubing. The coil has a capacity of about 1 ml. It is connected t o provide an optional parallel path between the pump and the column. The T-connections are constructed of Teflon and Nylon fittings as shon-n in Figure 2. Flow through the tubing is interrupted by spring clamps adjusted in tension for the pressure employed. The sections of tubing to mhich the clamps are applied are replaced from time to time as they become damaged. ,4t the beginning of a chromatogram, clamps 2, 3, 4, and 5 are closed. clamp 1 is opened, and the pump turned on. Eluent passes through the column and the sample coil is bypassed. After the base line registered by the recorder has become constant (about 1.5 hours), the coil is filled with the sample to be chromatographed. For this purpose clamps 4 and 5 are opened
