Detector Sensitivity

Another potential problem concerns the selection of the range of substrate concentrations to be used throughout the study. Considering the sensitivity of most HPLC detectors and the apparent Km values of most enzyme activities, the selection of the upper limit of concentration is usually not a problem. A problem will develop, however, when rate determinations are made at low substrate concentrations, since at these concentrations the amount of product formed during the course of the reaction will be small and may be below the monitor's level of detection.

Therefore, prior to executing any experimental protocols dealing with low substrate concentrations, it is prudent to determine what product concentra-

Resfrie tor loop

Column

Sample loading loop loading port

Sample

.plug

Sample loading loop

Chart marker

Figure 4.11 High-pressure injection valves. (A) The universal injector (Waters, inc.). A known volume of sample is injected, displacing a corresponding volume from the "sample loading loop" shown in lop diagram. After sample loading plug is replaced, the loading loop is placed "in line," and the eluent from pump moves sample onto column.

Chart marker

Sample plug

Figure 4.11 High-pressure injection valves. (A) The universal injector (Waters, inc.). A known volume of sample is injected, displacing a corresponding volume from the "sample loading loop" shown in lop diagram. After sample loading plug is replaced, the loading loop is placed "in line," and the eluent from pump moves sample onto column.

B

Model 7125 Schematic Diagram

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2

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J 3

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Needle Port

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Inject

Figure 4.11 (Continued) (B) The Rheodyne injector (Rheodyne, Inc.) in which the volume injected is determined by the sample loop. Sample loops can be obtained commercially in a number of fixed volumes or made to any size. (C) A 200-/U.L loop.

tions can be detected by ascertaining the lower limits of the detector being used.

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