Initial Rate Determination at Low Substrate

As in the case of any assay procedure, determination of the rate of product formation becomes difficult at the lower limits of substrate concentration. However, changes that can be made both in the assay system and in the chromatographic equipment can alleviate this problem. 

The first change is, of course, to increase the sensitivity of the detector. Most HPLC detectors contain range switches that make this a simple matter. When range switching is carried out, it is useful to determine whether calibration curves constructed at one range setting are still valid at another. 

the amount of product being detected may be increased by increasing the volume of the reaction mixture that is injected for analysis. With fixed-loop injectors this requires changing the loop, a rather simple procedure. If 

There is an upper limit to the volume of an injection, usually around 200 p,L. The injection of larger volumes results in spreading of the peak, a phenomenon that decreases resolution. However, it is possible to make loops of any volume and thus control this upper volume limit. If separation is adequate and resolution is not a problem, the loop volume may be increased. 

At times, obtaining enough reaction product requires the concentration of an entire reaction mixture. Following concentration, the residue is resuspended in a small volume of buffer and analyzed. 

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