507
Anal. Chem. 1986, 58,507-509 Lictr
Ha-Ne
Photodiode
Figure 1. Schematic diagram of the thermal lens densitometer: focusing lens 200 mm focal length; lens after the iris 100 mm focal
length.
k
56 z
tometer light source (25).The focused diameter is 0.061 mm, so excellent spatial resolution should be obtainable with minor modification of our instrument. Because the thermal lens densitometer detects the protein indirectly by measuring the binding dye, detection limits might be further reducted by more sensitive staining methods, such as silver staining. Nevertheless the enhancement of the sensitivity of Coomassie Brilliant Blue staining by thermal lens densitometer already provides clear advantages. First, the sensitivity of the simple Coomassie Brilliant Blue staining method can be made to match the sensitivity of the more complicated silver staining procedure, as measured with conventional instruments. Second, thermal lens detection of Coomassie Brilliant Blue stains provides senitive quantitation for proteins not amenable to silver staining (26-31). Extension of the thermal lens technique to quantitation of silver stains and exploration of other proteins is currently in progress. Registry No. Coomassie Brilliant Blue, 6104-58-1.
LITERATURE CITED (1) Sepanlak, M. J.; Vargo, J. D.; Kettler, C. N.; Maskarinec, M. P. Anal. Chem. 1984, 56, 1252-1257. (2) Leach, R. A,; Harris, J. M. J . Chromatogr. 1984, 218, 15-19. (3) Buffett, C. E.; Morrls, M. D. Anal. Chem. 1992, 54, 1174-1178. (4) Pang, T.-K. J.; Morris, M. D. Anal. Chem. 1984, 56, 1457-1459. (5) Collete, T. W.; Parekh, N. J.; Men, L. C.; Carreria, L. A,; Rogers, L. B. Pittsburgh Conference Abstract, 1985;056. (6) Chen, T. I.; Morris, M. D. Anal. Chem. 1984, 56, 19-21. (7) Chen, T. 1.; Morris, M. D. Anal. Chem. 1984, 56, 1674-1677. (8) Masujima, T.; Sharda, A. N.; Lloyd, L. B.; Harris, J. M.; Eyring, E. M. Anal. Chem. 1984, 5 6 , 2977-2979. (9) Peck, K.; Fotlou, K. F.; Morris, M. D. Anal. Chem. 1985, 5 7 ,
1359-1362. (IO) Fishbein, W. N. Anal. Biochem. 1972, 46, 388-401. (11) Fazekas de St. Groth, S.; Webster, R. G.;Datyner, A. Blochim. Biophys. Acta 1983, 31, 377-391. 3
4
5
6
DISTANCE FROM ANODE (cm)
Figure 2. Electrophoretogram scanned by the thermal lens technique: nondenaturing discontinuous electrophoresisof bovine serum albumin on 10% polyacrylamide gels with quantities as shown; insert, top to bottom, 6 ng, 4 ng, 3 ng of bovine serum albumin. The albumin dimer band is not shown.
the detection limit is area normalized to approximately 45 pg/mm2. This detection limit is similar to the silver-staining limit, reported as 20 pg/mm2 (24)to 42 pg/mm2 (15)in recent studies.
CONCLUSIONS We confirm the previous observations (19)that quantities of Coomassie Brilliant Blue stained bovine serum albumin less than about 125 ng are not visible to the eye. Nevertheless thermal lens densitometry performs well with these quantities of albumin. The dynamic range of the photothermal densitometer can be extended to higher concentrations than those studied here. Because of the nonlinear response of the Coomassie Brilliant Blue dye bipding and the saturation of thermal lens signals a t high absorbances, the useful range may not extend above a few micrograms. However, in the microgram region, the laser can be used directly as a conventional transmission densi-
(1s) Chrambach, A.; Relsfeld, R. A.; Wyckoff, M.; Zaccari, J. Anal. Biochem. 1967, 20, 150-154. (13) Switzer, R. C., 111; Merril, C. R.; Shifrin, S.Anal. Blochem. 1979, 98, 231-237. (14) Oakley, B. R.; Kirsch, D. R.; Morris, N. R. Anal. Blochem. 1980, 105, 361-363. (15) Morrlssey, J. H. Anal. Biochem. 1981, 117, 307-310. (16) Loening, U. E. Biochem. J. 1987, 102, 251-256. (17) Davis, B. T. Ann. N. Y. Acad. Sci. 1964, 121, 404. (18) Laemmli, U. K. Nature (London) 1970, 227, 680-685. (19) Reisner, A. H.; Nemes, P.; Bucholtz. C. Anal. Biochem. 1975, 64, 509-51 6. (20) Yang. Yen.; Hairrell, R. E. Anal. Chem. 1964, 56, 3002-3004. (21) Wallan. D. J. Ritz, G. P.; Morris, M. D. Appl. Spectrosc. 1977, 37, 475. (22) Sedmak, J. J.; Grossberg, S. E. Anal. Biochem. 1977, 79, 544-552. (23) Wilson, C. M. Anal. Biochem. 1979, 96, 263-278. (24) Merrll, C. R.; Goldman, D.; Van Keuren, M. L. Eiectrophoresis (Weinhelm, Fed. Repub. Ger.) 1982, 3 , 17. (25) Brayden, J. E.: Halpern, W. Anal. Biochem. 1983, 130, 9-13. (26) Wray, W.; Boulikas, T.; Wray, V. P.; Hancock, R. Anal. Biochem. 1981, 116, 197-203. (27) Friedman, R. D. Anal. Biochem. 1982, 126, 346-349. (28) Schleicher, M.; Watterson, D. M. Anal. Biochem. 1983, 731, 312-31 7. (29) Poehling, H.-M.; Neuhoff, V. Nectrophoresis (Weinheim, Fed. Repub, Ger) 1981, 2 , 141-147. (30) Guevara, J.; Johnston, D. A.; Ramagali, L. S.; Martin, B. A,; Capetillo, S.; Rodriguez, L. V. Electrophoresis (Weinheim, Fed. Repub. Ger .) 1982, 3 , 197-205. (31) Tsai, C.-M.; Frasch, C. E. Anal. Biochem. 1982, 119, 115-119.
RECEIVED for review July 17, 1985. Accepted September 19, 1985.
Versatile Instrument for Pulse Width Measurement Purnendu K. Dasgupta* and Ellis L. Loree Department of Chemistry, Texas Tech University, Lubbock, Texas 79409-4260
A variety of devices are available for measurement of fast pulse widths, oscilloscopes being the most common instruments used for measurements of pulse width lo00 s), the decimal points in all the display digits light via a 74LSOO inverter and an overflow status signal is also sent to defice E, which turns on the alarm and resets the start and stop flip-flops. Figure l a shows the necessary power supply. The assembled instrument is available from Berne TechLite Corp., Lubbock, TX.
RESULTS The performance of the counter was tested by sending rectangular wave pulses of variable duration (0.5-600 s) generated by a Shawnee digital electronic timer switch and a D cell. For any pulse duration at least 10 measurements were made and the counter was always found to be within one least significant digit (10 ms) of the period selected by the timer. For measurements with a flow injection system, the precision of repeated measurements was found to be 0.5%, which was the flow precision of the pump used, suggesting that the overall precision is controlled by the pumping system. Successive measurements could be made with the autoreset function as fast as the normal throughput rate of the system allowed. If throughput rate is too fast such that sample carryover occurs between successive injections, the counter is subject to the same errbrs as experienced by a conventional system. The counter is susceptible to false triggering if signal is accompanied by spurious noise (21mV), especially if the “start” and “stop” voltages are set to be the same. Such noise may be minimized by active filtering. LITERATURE CITED (1) Rhee, J.-S.; Dasgupta, P. K., submitted fot publication
RECEIVED for review April 18, 1985, Accepted September 9, 1985.
Continuous-Flow Injector for Flow Injection Analysis Amando F. Kapauan* a n d Marcelita C. Magno Department of Chemistry, Ateneo de Manila University, P.O. Box 154, Manila, Philippines Sample injection into the flowing carrier stream in flow injection analysis (FIA) is a weak link in this analytical technique. Manual syringe injection through an elastomer septum and rotary or sliding valve loop injection are the two most common methods currently used. The former is simple and inexpensive, but the reproducibity is dependent on operator skill, while the latter is expensive and has problems of its own (1). We have designed and fabricated a simple, inexpensive sample injector that controls fluid flow at the point where sample is pumped into the carrier stream, which is split be0003-2700/86/0358-0509$0 l .50/0
tween flow to the detector and flow to waste. A t no point during the injection cycle are any flow rates altered.
EXPERIMENTAL SECTION Sample Injector and FIA System, Figure 1shows the important details of the sample injector, while Figure 2 shows how it is incorporated into the FIA system. The sample injector body is acrylic (1/4-in.sheet stock) with one straight-through channel and two opposing side channels about 6 mm from each other. Short pieces of 18-gaugetubing from stainless-steel hypodermic syringe needles are epoxied on so that PVC tubing can be attached. The tubing labeled 0 in Figure 1 has an inner tubing, I (20-gauge 0 1986 American Chemical Society
