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1.4 QUANTITATIVE BIOCHEMICAL MEASUREMENTS

1.4.1 Analytical considerations and experimental error

Many biochemical investigations involve the quantitative determination of the con-

centration and/or amount of a particular component (theanalyte) present in a test

sample. For example, in studies of the mode of action of enzymes, trans-membrane

transport and cell signalling, the measurement of a particular reactant or product is

investigated as a function of a range of experimental conditions and the data used to

calculate kinetic or thermodynamic constants. These in turn are used to deduce details

of the mechanism of the biological process taking place. Irrespective of the experi-

mental rationale for undertaking such quantitative studies, all quantitative experi-

mental data must first be questioned and validated in order to give credibility to the

derived data and the conclusions that can be drawn from them. This is particularly

important in the field of clinical biochemistry in which quantitative measurements on

a patient’s blood and urine samples are used to aid a clinical diagnosis and monitor

the patient’s recovery from a particular disease. This requires that the experimental

data be assessed and confirmed as an acceptable estimate of the ‘true’ values by the

application of one or more standard statistical tests. Evidence of the validation of

quantitative data by the application of such tests is required by the editors of refereed

journals for the acceptance for publication of draft research papers. The following

sections will address the theoretical and practical considerations behind these

statistical tests.

Selecting an analytical method

The nature of the quantitative analysis to be carried out will require a decision to be

taken on the analytical technique to be employed. A variety of methods may be

capable of achieving the desired analysis and the decision to select one may depend

on a variety of issues. These include:

• the availability of specific pieces of apparatus;


• the precision, accuracy and detection limits of the competing methods;


• the precision, accuracy and detection limit acceptable for the particular analysis;


• the number of other compounds present in the sample that may interfere with the
  analysis;


• the potential cost of the method (particularly important for repetitive analysis);


• the possible hazards inherent in the method and the appropriate precautions needed to
  minimise risk;


• the published literature method of choice;


• personal preference.

The most common biochemical quantitative analytical methods are visible, ultraviolet

and fluorimetric spectrophotometry, chromatographic techniques such as HPLC and

GC coupled to spectrophotometry or mass spectrometry, ion-selective electrodes and

16 Basic principles