Peak area from chart recorder chromatogram

A technique for the quantitation of coloured bands or spots on chromatograms or electrophoretic strips. Colour intensity can be measured by transmission, when light is allowed to pass through the sample, or by reflectance, when light is reflected from the coloured area. TTie coloured areas are represented as peaks on a chart recorder, and, from this, the area under each peak can be determined. In some instruments, the area under each peak is automatically integrated. Albumin in some laboratories is measured by densitometric techniques. Densitometers can also be used in the measurement of amniotic fluid lecithin sphingomyelin ratios. 

The mixture is identical in each example. The peaks are shown separated by 2, 3, 4, 5 and 6 (a) and it is clear that a separation of 6a would appear to be ideal for accurate quantitative results. Such a resolution, however, will often require very high efficiencies which will be accompanied by very long analysis times. Furthermore, a separation of 6o is not necessary for accurate quantitative analysis. Even with manual measurements made directly on the chromatogram from a strip chart recorder, accurate quantitative results can be obtained with a separation of only 4a. That is to say that duplicate measurements of peak area or peak height should not differ by more than 2%. (A separation of 4a means that the distance between the maxima of the two peaks is equal to twice the peak widths). If the chromatographic data is acquired and processed by a computer, then with modem software, a separation of 4a is quite adequate. 

Weigh about 12 mg of barium chloride crystals into an empty sample tube and obtain chromatograms as indicated under Procedure. Repeat with 9. 6. 3 and 1 mg amounts of barium chloride crystals. Add the values for the two peaks obtained for water in each determination and correct for the blank. Construct a calibration graph relating milligrams of water to the corrected total peak height (or peak area) measured from the recorder chart. 

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