INSTRUMENTATIOIN BY RALPH H. MÜLLER
Optical Scanning Devices Offer Reliable Precise Results PTICAL SCANNING METHODS will con-
O tinue
to elicit t h e attention of analysts, particularly since t h e optical and electronic features of t h e technique a r e so precise and reliable. T h e limitations continue to be chemical in nature—i.e., preparation a n d uniformity of t h e sample. I n "Analytical Chemistry" 1962 which constitutes t h e proceedings of t h e international symposium held a t Birmingham University (U.K.), several authors discuss these points. (Elsevier Publishing Co. A m s t e r d a m / L o n d o n / N . Y., 1963; edited b y P . W . West, A . M . G. M a c donald, and T . S. W e s t ) . When a p plied to paper chromatograms, spot tests a n d rings formed in t h e Weisz ring oven system, difficulties arise which are definitely attributable t o nonuniformity of t h e spots or circles along with complexity of t h e matrix (paper or film). As H . Malissa concluded in his plenary lecture . . . " I n t h e field of qualitative analysis—organic and inorganic—spot analysis is an invaluable tool; for quantitative analysis the real bottleneck is t h e paper a n d further investigations a r e certainly r e quired for a full discussion of supporting materials." The careful a n d extensive work of Weis, Ottendorfer, a n d others h a s established these difficulties. Unfortunately, they have given rise to such statements as "comparing t h e visual measurements of t h e rings against instrumental density measurements, one finds t h a t mere visual comparison of the rings is a t least equal, if n o t superior, t o t h e instrumental measurements ! " This, a n d its implications, would n o t be believed b y a n y physicist or instrument specialist. Over t h e years, we have built dozens of scanners in which samples could be scanned repetitively and unattended. T h e scans were highly reproducible, even superimposable. Rarely were the peaks attractive or simple; in fact, t h e y delineated t h e i r regularity a n d random distribution in the sample in great detail. T h e elegance, simplicity, selectivity, a n d sensitivity of spot tests, chromatograms, a n d related tests make them very valuable. T h a t they can be made precise in a
quantitative sense a n d even more sensitive is a challenge which is not hampered b y instrumental resources a n d t h a t gets us right back to Malissa's concluding statement. Were this not true, then in colorimetric analysis wc would still be peering down through
Ncssler tubes. Such difficulties do not seem to be plaguing investigators working with electrophoretic patterns in gels. We wish to describe a new linear t r a n s port system for such evaluations. This relates to t h e Gilford Model 2410 Lin-
Figure 1 . Gilford Model 2 4 1 0 Linear Transport
Figure 2 . Scan of electrophoretic pattern of stained h u m a n s e r u m protein VOL. 40, NO. 11, SEPTEMBER 1968
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ANALYTICAL CHEMISTRY
See ACS Laboratory Guide for All Products/Sales Office
INSTRUMENTATION ear T r a n s p o r t made by Gilford I n s t r u ment Laboratories, Inc., Oberlin, Ohio 44074. Through the courtesy of S. M . Atkinson, we present some details of this device. This accessory will fit all Gilford monochromators, as well as Beckman D U monochromators equipped with Gilford photometers. This device, shown in Figure 1, moves electrophoretograms a t a constant rate through the measuring beam of a spectrophotometer. This results in a recording of absorbance as a function of lateral displacement. T h e sensitivity of the Gilford photometer allows the use of slits as small as 0.05 m m wide b y 2.36 m m high, to resolve very small bands, even though they are close together. This p h o tometer has a linear absorbance range of 0.000 to 3.000 Â, so t h a t more peaks can be kept on scale, even when scanning stained gels. T h e base line of the recorder can be offset u p to 2.900 A for increased sensitivity. Full scale sensitivities of u p to 0.100 A can be used with any base line u p to 2.900 A for extreme sensitivity to bands of low concentration. At present, the Model 2410 finds its greatest application in the scanning of polyacrylamide gels. T h e advantage of spectrophotometric rather t h a n densitometric analysis of these gels is t h a t a range of ultraviolet radiation can be used. This eliminates the need to stain gels and the accompanying lengthy destaining process. Also, there is no denaturation of the protein bands by stain, so proteins can be eluted from the gel and tested further. M u c h valuable information can be gleaned from a recording of absorbance vs. displacement. T h e exact position of bands in unstained material can be determined from the chart tracing. T h e height of the absorbance peaks shows the relative amounts of m a t e rial present in each band a n d the areas under the peaks can be obtained b y integration to determine absolute concentrations. Figure 2 shows a scan of the electrophoretic p a t t e r n of stained h u m a n serum protein. T h e gel was 7% acrylamide, 2.6% was linked with N,N'methylene-bis-acrylamide ( B I S ) . Here the albumin peak is off scale because its absorbance is about 3.95. This is readily accommodated b y range selection if all peaks are to be shown on one recording. Scanning was done a t 660 m m at a rate of 2.5 cm per minute. These are impressive results and the elaborate and intricate instrumentation is warranted. Few would want to "eyeball" a complex p a t t e r n like this.
