you probably may need to rewrite or verify your question. Plasma can not be 20 l in 800ul mixture

Dear Eslam,

I assume that we are looking at 20 microlitres of plasma.

I also assume that the molarity of each 800 microlitre aliquot must be:-

· 2.0 mM paraoxon(Diethyl paraoxon).

· 2.0 mM calcium chloride.

· 100 mM Tris or [2-Amino-2-(hydroxymethyl)-1,3-propanediol] -HCl buffer (pH 8.0).

If the above are the case, these are the calculations for making solutions:-

2.0 mM paraoxon:-

Molecular weight of paraoxon is 275.20 g.

1 M is 275.20 g/L.

1 mM is (275.20/1000) = 0.27520 g/L.

So 2 mM is 0.5504 g/L, or 0.5504 mg/mL.

In 800 µL, one would use (800/1000) x 0.5504 mg, i.e. 0.4403 mg/800 µL.

This is on the assumption that the paraoxon is pure.

Please check the purity of your paraoxon, the outside of its container should state the purity. If your paraoxon is not 100% pure, then the calculation for the actual amount to weigh out is (100/actual percentage purity) x the theoretical weight for 100% pure paraoxon.

However, weighing out 0.4403 mg/800 µL for each aliquot is tedious.

Hence it is simpler to make up a concentrated solution and add an aliquot of this concentrate to the final 800 µL solution.

For example, make up 100 mL of a pure paraoxon concentrate, using 550.4 mg/100 mL. This is a 20 mM solution.

A 1 in 10 dilution of this concentrate would be 2 mM.

So use a [(800/1000) x 0.1 mL] = 0.08 mL or 80 µL aliquot of the above pure paraoxon concentrate in the final 800 µL solution.

2.0 mM calcium chloride:-

Molecular weight of calcium chloride is 110.98 g.

1 M is 110.98 g/L.

1 mM is (110.98/1000) = 0.11098 g/L.

So 2 mM is 0.2220 g/L, or 0.2220 mg/mL.

In 800 µL, one would use (800/1000) x 0.2220 mg, i.e. 0.1776 mg/800 µL.

This is on the assumption that the calcium chloride is pure.

Please check the purity of your calcium chloride, the outside of its container should state the purity. If your calcium chloride is not 100% pure, then the calculation for the actual amount to weigh out is (100/actual percentage purity) x the theoretical weight for 100% pure calcium chloride.

However, weighing out 0. 1776 mg/800 µL for each aliquot is tedious.

Hence it is simpler to make up a concentrated solution and add an aliquot of this concentrate to the final 800 µL solution.

For example, make up 100 mL of a pure calcium chloride concentrate, using 222.0 mg/100 mL. This is a 20 mM solution.

A 1 in 10 dilution of this concentrate would be 2 mM.

So use a [(800/1000) x 0.1 mL] = 0.08 mL or 80 µL aliquot of the above pure calcium chloride concentrate in the final 800 µL solution.

100 mM Tris-HCl buffer (pH 8.0):-

Molecular weight of Tris is 121.14 g.

1 M is 121.14 g/L.

100 mM is (121.14/10) = 12.114 g/L.

This is on the assumption that the Tris is pure.

Please check the purity of your Tris, the outside of its container should state the purity. If your Tris is not 100% pure, then the calculation for the actual amount to weigh out is (100/actual percentage purity) x the theoretical weight for 100% pure Tris.

To produce 1L of 100 mM Tris, add 12.114 g of 100% pure Tris, to 800 mL of deionised water and mix well.

Adjust the pH to 8, using hydrochloric acid, then make up to 1L volume using deionised water and mix well.

However, in our analysis, the volumes of paraoxon, calcium chloride and plasma used are:-

80 microlitres plus 80 microlitres plus 20 microlitres , which equal 180 microlitres.

As the final aliquot volume must be 800 microlitres, the permissible volume of Tris used is (800-180) 620 microlitres.

So a [(800/620) or x 1.29 more concentrated Tris solution should be used in the final 800 µL solution.

Hence we need to produce 1L of 129 mM Tris, So add 15.6271 g of 100% pure Tris, to 800 mL of deionised water and mix well.

Adjust the pH to 8, using hydrochloric acid, then make up to 1L volume using deionised water and mix well.

Use a 620 microlitre aliquot of this Tris buffer to make up the final 800 µL aliquot solution.

Kind regards,

Ade Kujore

Marketing

Cecil Instruments Limited

Milton Technical Centre, Cambridge CB24 6AZ

United Kingdom

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Thanks... Ade Kujore