Measurement of Calcium
in DNA and DNP 1
The calcium content of aqueous solutions of deoxyribonucleic acid (DNA) and deoxyribonucleoprotein (DNP) cannot assayed directly because of interference effects. An acid extraction procedure enabled direct measurements of calcium in DNA and DNP, or a standard addition technique was used at higher concentrations.
Acid extracts are prepared by adding 1 volume of 1 M HCl to 19 volumes of sample solution containing DNA or DNP in water, and allowed to stand at 70°C overnight in a temperature-controlled hotbox in a sealed receptacle to avoid water loss. Where a suspension is present, the sample is centrifuged at 2000G for 10 minutes to obtain the soluble fraction. About 99% of the DNA and about 80% of the protein becomes soluble in HCl after standing overnight in HCl. The DNA concentration of DNP is approximately 40% w/w.
The sample solution should have a concentration of DNA between 50ppm and 100ppm DNA. Therefor it should be noted that readings may cause a lack of stability if ion concentrations are present at over 100ppm. Whilst the flame photometer is able to detect ion readings over 100ppm it is not recommended. This is due to matter which can be deposited in the nebuliser and effect further measurements by the droplets departing during other measurements. This means that the frequency that the nebuliser requires cleaning is increased and further dilution of the sample is recommended.
Calibration of the photometer
To calibrate the flame photometer a 1000ppm calcium stock solution is diluted into the concentrations of 10, 25, 50, 75 and 100ppm along with a 0ppm blank solution in 100ml volumetric flasks. These solutions were then aspirated into the flame photometer starting with the blank solution and working up in concentration.
Standard Addition Technique
The standard addition technique is a means of estimating the concentration in a solution where interfering metals are also known to be present.
1 Itzhaki, R., ‘Estimation of Calcium in Deoxyribonucleoprotein and Deoxyribonucleic Acid by Flame Photometry’, Anal. Biochem. 33, (1970), p.76-86