Determination of Beta-Carotene and Neo-BetaCarotene with the Visual Spectrophotometer F. P. ZSCHEILE AND B. W. BEADLE. Purdue University Agricultural Experiment Station, Lafaytttc, Ind.
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then studied on simpler instruments with a Type A-H4 mercury lamp, with clear standard glass bulb, a3 the light source. A Bausch & Lomb Universal visual spectrophotometer was employed, with and without the additional use of Corning flters 038 plus 511. The collimator slit was 0.25 mm. wide. The image of the line was well centered and masked on each side by the jaws of the exit slit.
HE relation of neo-/?-carotene to analytical methods for p-carotene has been discussed by Beadle and Zscheile ( I j , who successfully applied a photoelectric spectrophotometric method to the carotene analysis of certain vegetables. They showed that considerable error may be introduced into photometric methods for the analysis of carotene extracts jf the content of neo-&carotene is not considered. The 4500 A. region is often used for total carotene analysis by visual spectrophotometry. It is clear that if neo-/?-carotene is present, analytical results will be low, because neo-/?-carotene absorbs considerably less at this wave length than does pcarotene. In their method of analysis, a double monochromator is emloyed with the radiation from an incandescent source (T10 &ulb, C8 filament, 12-ampere, &volt projection lamp) of covtinuous roadiation. X%rrow spectrsl regions are isolated [ 10.5 A. at 4360 A. and 13.8 A. a t 4780 A.] at wave lengths which are optimum foroanalysisof mixtures of p-carotene and its neo-isomer. Thus 4360 A. is pmployed for determination of total carotene, after which 4780 A. is used for composition determination.
Results obtained by two operators are compared in Table
I with the result obtained on the more accurate photoelectric spectrophotometer. Each visual determination is the average of 10 observations made in rapid succession. In an effort to obtain greater precision and speed, a modified KWSZ flter photometer (Wilkens-Anderson Co., Chicago, Ill.) was employed with special gas-filled photocells having potassiumcoated cathodes to obtain maximum sensitivity in the blue regiop. The following combinations of filters were tried: for 4398 A. Corning 038 plus 511, and for 4916 A. Corning 555 plus GG 11 and Corning 555 plus 556 plus GG 11 (Fish-Schurman glass filter). Analyses were also made with the visual instrument and incandescent light source (1000-watt projection lamp with T 20 bulb), using wave lengths employed by Beadle and Zscheile ( 1 ) . Slit 1 was 0.20 mm. wide and slit 2 was 0.15 mm. wide at 4360 A. and 0.7 divisions wide at 4780 and 4850 A. Results for total carotene were in error by only 2.5 per cent. Analyses for composition had greater errors, although averages for the two wave lengths were in good agreement with correct values. The mercury arc source is a distinct advantage for this type of visual measurement.
Since the type of optical equipment employed b y Beadle and Zscheile is not generally available, it is desirable that this analytical method be adapted to simpler and less expensive instruments. It is evident (Figure 1,1j that certain lines from the mercury arc are favorably situated in the spectrum to permit their use in this analysis. This paper presents the absorption constants required for the use of a mercury arc source with any optical instrument capable of isolating the 4358 and 4916 A. lines and with a photometer of suitable sensitivity. Comparisons are made between the use of a visual spectrophotometer and of a photoelectric filter photometoer. Among$he mercury lines in the region of 3800 to 5200 A., the 4358 A. line is most fortunately situated, because at this wave length the absorption curves for the two carotenes are coincident. [In such a region, where the slopes of both standard curves are not extremely steep and absorption coefficient: are high, theopercentage difference between values at4360 A. (1) and 4358A. is negligible.] The 43393nd 4347 A. lines are consideroably weaker than the 4358 A. line, are close to the 4358 A. line, and occur where the absorption coefficients of the two carotenes are nearly identical. The fact that the slopes of the absorption curves of both compounds are steep at 4916 A. is not objectionable because a line source is used. Another line, of approximately equal intensity, occurs a t 4960 A., the inclusion of which would very seriously affect the composition determination. The pzoblem is thus resolved into a n Sffective isolation of the 4916 A. line and the group near 4358 A.
TABLE11. ANALYSES OF FRESHSPINACH WITH VISWALAND PHOTOELECTRIC SPECTROPHOTOMETERS
Wave Length
A.
4916 4358
4358
1.0 0.5 0.5
--
Total Carotene Visual Operator Operator 1 2 Photoelectric MicroMicrooramslg. gramalg. Micrograma/O. 52.6 52.6 52.6
54.9 56.7 56.7
55.6
... ...
Composition (% ,+Carotene) 4916
None
1.0 0.5
%
%
93.5 91.5
94.5 96.5
% 8 8 . 5 (4780
...
A,)
Discussion The 4358 A. line is very strong and easily used in the visual method with a filter combination of Corning glass filters 038 and 511. This filter i s g o t necessary, however, as shown by the results. The 4916 A. line is not strong and should not be filtered. Care must be taken t o limit the beam to this line only and t o exclude the 4960 A. line. The results of these two operators disagree appreciably, apparently for subjective reasons, but agreement is better at Io higher concentrations (log = 0.5 to 0.7 for relative concentration of 1.Oj. The agreement between the visual and photoelectric spectrophotometric methods is satisfactory for many applications. For composition determination, differences were 3 to 9 per cent, permitting indication of relatively large amounts of isomerization. The experience of both operators with the visual instrument was very limited. It is probable that a more experienced operator would obtain results more concordant with those from the photoelectric spectrophoto-
Specific Absorption Coe5cients in Hexane Solution 8-Carotene Neo-p-Carotene 142.5 196
None Present None
A.
TABLEI. ANALYTICAL CONSTANTS Wave Length
Filter
Relative Concentration of Care tene
42.5 196
Experimental Table I presents the analytical constants as numerical values from the graphic spectra reported earlier (1). Solutions from fresh spinach extracts were analyzed as standards by the method of Beadle and Zscheile. These solutions were 633
I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY
634
metric method. Some scattered light was no doubt present in the radiation employed in the above visual measurements, Instrument design and quality of optical parts are important factors. With the photoelectric filter photometer, absorption values obtained were much too low, indicating that other lines were not sufficiently removed by the filter combinations. It was concluded that the group of lines near 4358 A. was not completely isolated by the filters available and that such filters alone are inadequate to isolate the 4916 A. line. These filters are thus inadequate to permit an analysis of this carotenoid mixture with a photoelectric filter photometer. These analytical constants and this limited experience with the visual method are presented in the hope that they may be useful to workers employing visual equipment. It is noted that Equation 4 (1) fcr calculation of per cent /3-carotene is simplified for A4916 A. because the denominator becomes 100, the difference between the specific absorption coefficients. It is highly probable that the new types of photoelectric spectrophotometers, which have recently appeared on the
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market at comparatively low cost and which employ a mercury arc source, would isolate these mercury lines sufficiently well and also provide greater precision and speed than are possible with the visual method.
Summary &carotene and neo-p-carotene can be determined in properly purified solutions from spinach extracts by means of a visual spectrophotometer, preferably with a Type A-H4 mercury lamp. A photoelectric filter photometer did not isolate sufficiently narrow spectral regions for accurate analysis of this carotenoid mixture. Tke total carotene is calculated from the absorption at 4358 A. and the percentage of either carotene from the absorptions a t 4358 and 4916 A.
Literature Cited (1) Beadle, B. W., and Zscheile, F. P., J. Bid. Chem. 144, 21 (1942). JOURNAL
Pager 6, Purdue University Agricultural Experiment Station.
This research was supported in part by a General Foods Corporation Fellowship.
Pipet-Type Capillary Viscometer For Substances Which Are Solid or Highly Viscous at Room Temperature J. F. WEILER, Coal Research Laboratory, Carnegie Institute of Technology, Pittsburgh, Penna.
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HIS viscometer was developed for routine determina-
tions of the kinematic viscosities of the heavier products resulting from the destructive hydrogenation of coal, These products have a low viscosity index and, although sufficiently fluid at 98.89' C. (210' F.) to permit the use of a capillary viscometer, are either solid or highly viscous at and considerably above room temperature, so that the charging of an Ostwald viscometer is difficult. The several modifications of the U-tube viscometer having automatic or nonautomatic accurate charging features have r e c e n t l y b e e n briefly discussed (3). However, for ease of charging and cleaning, size of sample required, and recovery of the sample, none is as convenient as the pipet-type viscometer such as described by the Bureau of Standards ( 2 ) . A serious disadvantage of this instrument is that it requires a special constant-temperature bath from which it cannot be removed without disturbing the bath, The so-called Bureau of Mines pipet viscometer (1) can be handled in the same manner as the Ostwald type, in that several viscometers may be installed in the same bath and can be removed and replaced without disturbing the bath. The pipet, however, must be charged while out of W CAP the bath. This limitation not only causes difficulty in charging materials which do not flow readily a t room temperature, b u t also necessitates removal
of the pipet for recharging for a check determination. The viscometer described in this communication makes use of a novel method of handling the sample to secure the advantages of both of the above cited modifications of the pipet viscometer. METHODOF USE. The sample (5 to 10 cc.) is contained in a sample vial of 15-cc. capacity having a screw cap. With the viscometer removed from the bath, vessel 4 is detached at the ground joint and the sample vial is screwed into the sample mal cap which is suspended b a thread leading up through support arm B of the viscometer. T i e crown of this cap has been cut out,.sa that the pipet tip can pass through it. A is fastened in place with springs and the viscometer is placed in the bath. A small plumb bob, suspended in the support arm, is useful in securing vertical mounting of the viscometer. When the sample has become fluid, the vial is drawn upward until the pipet tip touches or nearly touches the bottom of the vial. A metal disk serves as a counterweight t o hold the vial in this position. When the sample and viscometer have attained bath temperature, the sample is sucked up into the pipet to the correct charging level and the vial is then lowered to the bottom of A , in which position the pipet tip will clear the top of the liquid in the vial after the contents of the pipet have been discharged, The time required to discharge the pipet can now be determined. Any number of repeat determinations can be made without dismantling the pipet, After the determinations the sample drains into the vial and is available for other uses with a minimum of loss. Results are reproducible to 0.2 per cent. If the dimensions of the capillary and bulb of the viscometers of this design are made t o correspond to those of the Ostwald-Fenske A. S. T. M. viscometers, these viscometers will be about twice as fast, owing to the larger head of liquid. These viscometers have a record of successful use in the author's own laboratory. Uncalibrated viscometers t o accommodate a screw-cap vial 8 cm. in length by 2 em. in diameter and wlth capillaries and bulbs of the same dimensions as the OstwaldFenske A. S. T. M. viscometers were obtained from the Scientific Glass Apparatus Company.
Literature Cited (1) Dean, E. W., Hill, H. H., Smith, N. A. C., and Jacobs, W. A., U. S. Bur. Mines, Bull. 207, 45 (1922). (2) Mair, B. J., Schicktanz, S. T., and Rose, F. W., Jr., J. Research Natl. Bur. Standards, 15, 557-74 (1935). (3) Ruh, E. L., Walker, R. W., and Dean, E. W., IND.ENG.CHEX., ANAL.ED.,13, 347 (1941).
