Sample
Sample size/volume
Preservative
Stability
Comments
Water
50 ml
0.2 g ethylene dinitrilo-tetraacetic acid (EDTA), disodium salt
2 weeks when stored at 4° C. Up to one month or more if kept at −20 °C
Remove coarse particles by simple filtration/sedimentation
Urine
50 ml midstream urine collected preferably during morning hours
0.2 g EDTA,
disodium salt
2 weeks when stored at 4 °C. Up to one month or more if kept at −20° C
As above
Blood
20 ml blood/plasma/serum
Heparin at the rate of 20 I.U. per ml blood
Plasma/serum samples can be stored for 2 weeks at 4 °C and up to one month or more at −20 °C
Plasma/serum is better than whole blood for storage
Bone
A piece of flat or long bone (approximately 10 g)
Remove flesh, dry and convert into ash after alkali fusion
Bone ash can be preserved up to one month or more
Muscle and fat layer is removed carefully
Fodder
Collect at least 20 samples (each equivalent to 100 gm dry matter) and pool them to make one composite sample
Weigh the fodder and dry in shed. Can also be dried in oven at 70–80 °C for 24–48 h
Can be stored at 4 °C for a month
See the guidelines of centre d’expertiseen analyse environnementale du Québec*
Feed/feed-supplement/mineral mixture
Collect 20 representative samples randomly, each about 100 g and mix them properly to make a composite sample
Take 100 gm composite sample. No added preservative is required
Can be stored for several months at room temperature
Ashing may be essential to remove organic material
Egg
One whole egg
No added preservative is required
2 weeks when stored at 4 °C
6.5.4 Electrode Preparation
The fluoride selective electrode is filled with electrode filling solution (supplied/recommended by the electrode manufacturing company) before fluoride analysis. After filling the solution up to the recommended level, hold the electrode body with one hand and push down the electrode cap with the thumb to drain out one to two drops of the filling solution. The level of electrode filling solution during F analysis should be at least one inch above the level of the sample. Care should be taken that the O-ring of the electrode is moistened before estimation by tilting the electrode, and the fill hole should always remain open during the analysis. The ISE is now connected with the meter through the cable and the meter is switched on.
6.5.5 Checking Electrode Operation
Electrode operation can be checked by measuring the slope of the electrode. Electrode slope is defined as the change in millivolts observed with every tenfold change in fluoride concentration. For checking electrode operation, the meter is set in mV (millivolt) mode. Take a beaker and place 100 ml mixture of TISAB and deionized water solution in a suitable ratio. Add the fluoride standard to adjust the fluoride concentration of the solution 1 ppm. Take the reading in mV. Again take 100 ml mixture of TISAB and deionized water solution and add the fluoride standard solution to adjust the fluoride concentration of the final solution 10 ppm. Record the mV reading. There should be a 54–60 mV difference between the two readings when the solution temperature is between 20 and 25 °C.
6.5.6 Preparation of Standards
Place sodium fluoride powder at 110 °C for 2 h in a desiccator before weighing. Take 2.21 g dried sodium fluoride and dissolve in one liter deionized water. This will give a stock standard solution with 1000 ppm fluoride concentration. A range of standards of decreasing concentration are prepared by serial dilution of the stock standard solution. The concentration range of standards used for calibration of the instrument must be wide enough to accommodate the expected fluoride concentration in the test solution (sample). Readymade fluoride stock standard solutions are also available on the market.
6.5.7 Analytical Techniques
Several analytical techniques can be used for fluoride analysis using ISE. The selection of a technique depends upon sample volume, expected fluoride concentration in the sample, chemical properties of the sample solution, and individual choice. A brief description of important analytical techniques is given below.
6.5.7.1 Direct Calibration
In this technique, the instrument is calibrated using a series of standard fluoride solutions, and thereafter F in the test sample is analyzed directly. This technique is suitable when the fluoride concentration in the test sample is fairly high, a large variation in F is expected, or rapid analysis of a large number of samples is required. Both standards and test solutions are mixed with TISAB to make their ionic strength equal. Temperature of the standards and test solutions should be nearly equal and the difference between temperatures should not exceed 2 °C. In this method, the volume of the sample taken has no influence on the measurement. Concentration of fluoride in the test solution is expressed as ppm or moles per liter (one mole per liter fluoride is equivalent to 19,000 ppm). Details about calibration of the instrument are available in the literature (Buck and Cosofret 1993). It is recommended to prepare and use four standards for calibration of the instrument. Each standard should have ten times less fluoride concentration than the last. For example, standards containing 1000, 100, 10, and 1 ppm fluoride may be used for calibration. Readings can be taken in mV or directly in concentration mode.
6.5.7.2 Incremental Techniques
Incremental techniques are best suited when complexing or interfering ions in the test sample are present in excess (50–100 times the test ion, i.e., fluoride ion). The instrument can be run in mV or in concentration mode. Incremental techniques can be further classified into the following categories:
(i)
Known Addition
This technique is useful for test samples containing low fluoride concentration, checking results of the direct calibration, or fluoride estimation in samples containing a high concentration of interfering ions. Here, the electrode is immersed in the sample and TISAB mixture and an aliquot of fluoride standard is added. The fluoride concentration in the test sample is determined by calculating the difference in readings before and after addition of the fluoride standard solution.
(ii)
Known Subtraction
This technique is based upon titration and utilizes the stable standard of any species that reacts completely with the test ions (fluoride) present in the solution. Here, standards of the reacting species (ions) are added to the test sample and the difference in reading before and after addition is calculated. In this technique, knowledge of the stoichiometric ratio between the standard (reacting ion) and sample (test ion/fluoride) is necessary.
(iii)
Analate Addition
The electrode is immersed in a mixture of fluoride standard solution and TISAB and a measured quantity/volume of the test solution is added. The difference in readings before and after addition of the test solution is calculated and the fluoride concentration is determined. This technique is useful for determining fluoride concentration in soluble solids or powders, viscous samples, samples with high fluoride concentration, or when interfering ions are present in high concentration in the sample. However, it is not suitable for samples having a low fluoride concentration.
(iv)
Analate Subtraction
This method is used for analysis of those ions for which ion selective electrodes are not available. For example, a fluoride selective electrode can also be used for analysis of aluminum ions in the solution by detecting the amount of sodium fluoride required to react completely with all aluminum ions present in the solution. Aluminum reacts with fluoride ions to form aluminum fluoride, which is insoluble in water.
(v)
Titration
In titration techniques, an ion selective electrode is used just as a litmus or universal indicator for determining the end point of the titration. The advantage of using an ion selective electrode is an increase in the accuracy of results, although it is a time-consuming cumbersome process. This technique is seldom used for fluoride analysis.
6.5.8 Fluoride in Acid Solution
The pH of the acidic solutions (pH below 5) must be adjusted into a weakly acidic to a weakly basic range before fluoride analysis. Addition of a strong base such as sodium hydroxide is not recommended for pH adjustment because the total ionic strength of the adjusted samples and standards will vary with the amount of added sodium hydroxide. A sodium acetate solution (15 %) can be used for adjusting the pH of acidic solutions.
Procedure
1.
Prepare 15 % sodium acetate solution in deionized water.
2.
Prepare a background solution containing all components of the test solution except fluoride.
3.
Prepare standards in the concentration range of the test samples by adding fluoride to the background solution. Dilute each standard tenfold with the sodium acetate solution (9 parts sodium acetate and 1 part standard).
Stay updated, free articles. Join our Telegram channel
Full access? Get Clinical Tree
