MICROSPECTROSCOPY AND MICROSPFXTROPHOTOMETRY Ji 257 



length. Provided Beer's law be applicable, then: 



d, = KIC. (10.4) 



Measuring the concentration C requires, therefore, measuring the 

 thickness / of the object. Such measurement is not always easy to 

 carry out and it is more convenient to substitute it for an area measure- 

 ment thus leading to the determination of the mass of the substance 

 in the object, instead of concentration. 



Let M be the mass of the substance in the object and s its surface, 

 then: 



M 



C = ^ (10.5) 



si 



whence 



sd, 

 M =-^- (10.6) 



A 



Provided A", at the wave-length considered, be known and having 

 measured d-, and the area s of the object, the mass M of the substance 

 in the object is educed. Equation (10.6) shows that, when small 

 quantities of substance are to be measured, the areas must be small too. 

 In an area of 1 /r, approximately 5 x 10^" gr of nucleic acid may be 

 detected (A' = 20 at 2600 A). These numerical values correspond 

 to d, = 01. 



In this way, Thorell measured the quantity of haemoglobin in 

 a single living cell. The curve in Fig. 10.15 shows the changes in 

 absorption 1 — ///o (/q = incident intensity, / = intensity transmitted 

 after passing through the object) along the line ox. Absorption at 

 any point of the object can be determined by scanning. Once the 

 shape and dimensions of the cell are known, the haemoglobin con- 

 centration at any point of the object can then be calculated. The 

 aggregate quantity M of haemoglobin is eventually computed by 

 integrating the absorption curve over the whole area S of the cell. 

 According to equation (10.6), then: 



M =jj'^dxdy. (10.7) 



.9 



Equation (10.6) also shows that there is no need to determine K 

 if all that is required is to compare the masses Mi and M2 of the same 

 substance in two different objects. This is the problem which arises 



