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THE HPLC ADAPTOR
tem, the increase in fluorescence is proportional to the amount of lactate, in contrast to the small decrease in fluorescence used in the ammonium assay. An advantage of working with these small samples is that surface tension can sometimes be exploited effective ly. In the case of the fluorometer, the sample is injected directly into the re agent stream without going through a valve or seal. The injection port is merely a small hole in the side of the tubing carrying the reagent from the reservoir to the cuvette. As with many of these instruments, the sample pipet is mounted in a holder attached to a binocular microscope so that the user can always see the pipet tip and place it precisely in the middle of the re agent stream. Reagent flow rate for the ammonium assay is about 4 /zL/min and 1 ^L/min for lactate, so reagent consumption is not an eco nomic factor.
(a) Form bend and add glass.
(b) Blow very thin bubble.
Colorimetry
IT'S UNIVERSAL . . . adjusts to all HPLC Columns IT'S HAND-TIGHT . . . seals to 5,000 PSIG IT'S DOUBLE-SIDED . .. adapts to male and female fittings IT'S HASSLE FREE . . . connects without wrenches
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While fluorometry can have high sensitivity and can be adapted to mea surement of many materials, colorimetric procedures often are simpler. We have constructed a flow-through colorimeter with a working volume of 0.3 μι, and a path length of 1.1 cm. Two techniques made this feasible. One was to make the optical windows from the capillary which formed both the cuvette and connecting tubes. The other was to coat the outside of the tube with a light-absorbing substance. This step prevents light that travels in the glass, bypassing the sample, from reaching the photosensor. Although it is difficult to attach flat windows to small capillaries, acceptable windows can be formed by a modified glassworking technique. Using a stereo microscope and ei ther a miniature gas-oxygen torch (for quartz) or a hot Pt-Ir wire (for soft glass), two right-angle bends are formed about 1 cm apart (17). A bit of extra glass is added to the bend, and it is blown out to a thin bubble (Figure 4). With careful heating, the bubble is allowed to collapse, forming a reason able window at the end of the cuvette. The imperfection of the windows makes each cuvette different from one another, but each can be calibrated. The effect of fluid refractive index changes must be measured for this sort of cuvette just as for other flowthrough cuvettes. Although the first colorimeter of this series used an in jection port similar to the CO2 analyz er, it became apparent that a hole in the side of the lead-in tubing was sim pler and eliminated the possibility of mercury contamination. Single-wave length colorimeters have been devel oped for measurement of Mg 2+ ,
CIRCLE 1 ON READER SERVICE CARD
398 A · ANALYTICAL CHEMISTRY, VOL. 56, NO. 3, MARCH 1984
(c) Collapse bubble to form nearly flat window. Figure 4. Construction of quartz capil lary cuvette The final product has two 90-degree bends con structed as follows: The capillary is sealed at one end and connected to a 10-mL glass syringe at the other to allow the intraluminal pressure to be increased, (a) Beginning with the capillary hori zontal, the initial bend is made by heating a small section of the capillary while maintaining gentle pressure to keep the lumen open. An additional small amount of quartz is melted onto the bend to thicken the wall, (b) The quartz is then softened with the flame, and a bubble is blown at the bend. (c) The portion of the bubble that is to be in the light path is flattened by local application of heat and gradual reduction of intraluminal pressure
PO4 3 -, Ca 2+ , and urea. Recently, a system with an adjustable wavelength source has been constructed so that the colorimeter can be adapted quick ly to different methods (18). These miniature flow systems re quire very constant reagent pumping rates. We have used synchronous motor-driven syringes to draw the re agent through the cuvettes from the reservoirs. Flow variations as small as 1% peak to peak are detectable, so the screws and gears of the pump drive must have good precision. An alterna tive to the pump would be to use a capillary to control the flow generated (continued on p. 405 A)
