A simple, inexpensive spectrophotometer accessory for use in microanalytical studies

ANALYTICAL

BIOCHEMISTRY

60,

617-620

(1974)

SHORT COMMUNICATIONS

A Simple,

Inexpensive Spectrophotometer Accessory for Use in Microanalytical Studies1

The presence of compounds in small volumes of starting materials often poses problems with regard to their ultimate detection by spectrophotometry. Several designs of microcuvettes have been reported (1,2) and a variety are available commercially. However, they (a) are normally expensive, (b) are restricted in volume and path length and hence are often useful only for specialized reactions, (c) often require elaborate positioning devices, and (d) require care in filling and cleaning and are usually not disposable. The potential of fiber optic material for spectrophotometric work has already been recognized by Vurek and Bowman (3). In this report we describe a spectrophotometer accessory which utilizes fiber optics to eliminate the disadvantages described above, which are associated with the use of conventional microcuvettes. The use of fiber optics allows flexibility in the system as it means that a microcuvette can be positioned in an easily accessible location away from the normal cell compartment. Moreover, in place of expensive, specialized cuvettes, disposable capillary tubes such as the readily available microhaematacrit capillary tubes (Corning Glassware) which hold approximately 80 ~1 can be used. Unlike the normal spectrophotometric cuvettes, fingerprints on the external surface of the capillary will not affect the transmission of light. Because the microcell holders themselves are freely moveable, path lengths between 0.5 and 6.0 cm can be obtained with this system. Construction of the accessory. The dimensions of the accessory are based on the cell compartment of a Shimadzu QV50 spectrophotometer which is of similar dimensions to a Beckmann DU, Gilford 240 or Unicam SP5OO. Obviously, however, any model spectrophotometer possessing a suitable photomultiplier to ensure a high degree of sensitivity could be used and the dimensions of the accessory adjusted accordingly. ‘Supported c opyright &411 rights

by

a National

Health

@ 1974 by Academic Press, of reproduction in any form

and

Medical

617 Inc. reserved.

Research

Grant.

618

SHORT

COMMUNICATIONS

Basically, all that is required is a light-proof box to house the microcell holders, a rigid holder to position the fibers in the spectrophotometer cell compartment, and two short lengths of fiber optics (Dolan and Jenner Industries, Melrose, Mass.). The accessory simply positions directly into the cell compartment and is removed at any time when standard cuvettes are required. The accessory (Fig. la) was constructed out of 3 mm thick black Perspex sheet (I.C.I., acrylic plastic) with the exception of the micro holders which were fashioned from lo-mm Perspex rod. All joints were cemented with Tensol No. 1 liquid cement (I.C.I. Melbourne, Australia). The accessory comprised a base piece (150 X 150 mm) to which a light-tight box was attached on the top. If the fit of the lid is not perfect, any stray light can be excluded by gluing black felt in position. The lid of the box was hinged with stainless-steel hinges. Beneath the base piece, another light-tight margin was formed so that it fitted snugly into the spectrophotometer cell compartment. A single

LIGHT

FIBER

;d CAPlLLyY -/IC FNOM BRASS

-10

LlQHTw SCREWS

(6)

OPTICS

_. FIBER

PM y3a

TUBE

%

BOX

,a. Ibl

BRIDGE

t HOLDER WITH SET SCREW

FIG. 1. (a). Diagram (to scale) of the accessory illustrating the microcell holders positioned in the light-tight box with the fiber optic support plate projected beneath the base plate, (b) inset (actual size) of the fiber optic support plate from an end view, (c) inset (actual size) of the microcell holders.

SHORT

COMMUNICATIONS

619

piece of Perspex was cemented to the base piece in the center of this area to support the fibers in line with the light source and the photomultiplier (Fig. lb). Exact alignment is not absolutely necessary in this system, but it is an obvious advantage. TWO small plates of Perspex (20 x 20 mm) grooved on the inner surface and with a central hole, were used to hold the fibers rigidly in position. One end of each fiber was bent to a 90” angle by placing it in close proximity to the tip of a hot soldering iron. The ends were then located in the holes and the plates screwed to the Perspex support. The simplicity of mounting the fibers enables a rapid changeover to any other size of fiber which might be required and also the easy replacement of a damaged fiber. The microcell holders (Fig. lc) were constructed to hold the fibers for easy alignment with the capillary tube and also to support the capillary. A center hole (diam 1 mm) was drilled through the Perspex rod and then a half-section was cut away as illustrated in the diagram. The cutaway in the holder permitted easy rinsing of the fibers after use. The microcell holders were retained between guide bars so that any desired variation in path light could ‘be readily obtained, one holder being held in position with a set screw. The bridge in the middle gave support to the capillary and enabled easy placement of the capillary into the cell holders. At any fixed path length, absorbance measurements were always reproducible. The fibers were encased in polyamide tubing (ID 0.75 mm, OD 0.94 mm) to give rigidity to the fibers when they were held in place by the retaining screws in the microholders and also to eliminate damage or contamination to the ends of the fibers. The fibers themselves were of a diameter (approximately 0.70 mm) to just fit inside the microhaematacrit tube. As the size of fiber available is quite extensive, the choice of capillary for use in the system can also be varied. The thicker walled capillary tubes are to be preferred, as reproducibility is affected if bending of the capillary tube occurs. A lens was formed on each end of the fibers by warming near a hot iron and this ensured a more coneentrated beam of light. The formation of the lens and the sealing of the polyamide was accomplished at the same time. The plastic optical monofibers described in this report have been used satisfactorily for absorbance measurements in the visible range from 400-660 nm of a wide variety of colored solutions. Figure 2 illustrates a typical miCrOanalySiS of solutions of p-nitrophenol in 0.02 M NaOH using the accessory. As the fiber optics themselves are encased in polyamide tubing, measurements are possible in mild alkaline and acid medium and in most organic solvents. For measurement in the ultraviolet range, it should be possible to sub-

DZU

SHORT

COMME’iIC.~TIO?iB

“g

P-NITROPHENOL

/01

ML

FIG. 2. Absorbance measurement at 410 nm of solutions using microcapillary tubes with a 6-cm light path.

of p-nitrophenol

stitute quartz optical fibers for the plastic fibers. The difficulty of obtaining quartz fibers, however, has precluded investigations of this aspect. The advantage of this accessory over others lies in the inexpensive nature of the materials used in its construction and the ease of construction and operation. The versatility of the system in respect to the size of the microcapillary used coupled with the adaptability of the system to long or short path-length measuremnts makes the accessory ideal for use in microanalytical studies.

1.

REFERENCES GLICK, D. (1963) Quantitative Chemical Techniques of Histo and Cytochemistry, Vol. II, p. 17, Interscience,New York.

2. HOWELL, D. S., PITA, J. C., AND MAWJEZ, J. F. (1966) Anal. Chem. 34 3. VUREK, G. G., AND BOWMAN, R. L. (1969) Anal. Biochem. 29, 238-247.

434438.

J. D. SALLIS J. E. JOFLDAN Department

of Biochemistry University of Tasmania Hobart, Tasmania, Australia Received January 8, 19?‘4; accepted

February

Z?O, 1974