Version 12-22-16

Using Hamilton Syringes:

Use a Hamilton syringe to pipet organic solvents accurately. For example you cannot pipet small vols (5-10 uL) of chloroform with a Pipetman.

Step 1: First you need to be sure the syringe is clean and that it is charged with the solvent that you will use as your dilution solvent (i.e. methanol). Take a glass tube with about 1 mL of methanol in it and pump the plunger up and down 4-5 times, then with the plunger full down, transfer the syringe to a second tube with ~1 mL methanol and repeat. Finally do this with a third tube of ~1 mL of methanol. Be sure the 3 tubes are labeled 1,2, and 3 and rinse in order, in this way the impurities tend to be mainly in tube 1, less in tube 2 and even less in tube 3.

Step 2: Now you need to charge the clean syringe with the solution you wish to take a small volume from. You need to be sure the needle and syringe barrel are well rinsed with this solution so insert the syringe into this solution and pump up and down 4-5 times. The volume of solution in the tube should be at least 100-200 uL so that addition of the solvent left in the needle after rinsing (step 1) is insignificant. The volume in the needle is < 1 uL and so the volume will go up from say 100 to <101 uL, not much. Then draw up solvent to the desired point on the syringe barrel and be sure there is no trapped air in the barrel between the bottom of the plunger and the bottom of the syringe, check carefully.

Step 3: Remove the syringe from the solution in step 2 and place the needle in the tube containing the dilution solvent, say 100-200 uL, push the syringe once to the fully down position and remove the syringe. Do not pump up and down because you only want to deliver the solution in the barrel of the syringe, not what is left in the needle.

Step 4: If you want to make an even more diluted solution you have to repeat steps 2 and 3 above. So first rinse the syringe in the once diluted solution by pumping up and down a few times, then draw up the volume of diluted solution you want and transfer the solution to the new tube with at least 100-200 uL of pure solvent in it. Press the plunger down all the way but dont’ pump up and down.

Just think carefully: To rinse the syringe you have to rinse the needle and the barrel and so you have to pump up and down, and you want the volume of this solution to be big enough so that rinsing with the syringe with pure solvent in the needle will not increase the solution volume much. When you deliver the solution to the new tube of solvent you want to deliver only the barrel volume and not what is left in the needle so you should not pump up and down at this stage. But if you make a further dilution you have to bring the syringe to a state of rinsed well with the solution you want to dilute (pump up and down) and then deliver the barrel volume only to a tube of pure solvent so you push the needle down once without pumping up and down.

As an example, suppose you want to make a solution of 0.00025 uL of DMF per uL of CD3OD (for doing quantitative NMR where DMF is the internal standard). So you take 3 tubes of pure DMF, each with about 1 mL of DMF, and rinse the syringe as in step 1. You then draw up 5 ul of DMF from tube 3 of step 1 and check for trapped air, if you have air, displace the DMF and draw again. You then put the needle into a tube with 195 uL of CD3OH and discharge the 5 ul of DMF but don’t pump up and down. Now you have a solution of 5 ul of DMF in 200 uL of total solution, which is 0.025 uL DMF per uL of methanol, but you want 0.00025 uL DMF per uL of methanol, so you have to further dilute 100-fold. So you put the syringe into the 0.025 uL DMF per uL CDOD solution and pump up and down a few times and draw up 2 ul of solution and transfer it to a new tube with 200 uL of CD3OH and push the plunger down all the way without pumping up and down.

If you want to deliver 2-10 uL, use a 10 uL syringe (avoid drawing up less than 2 uL into this syringe), if you want to deliver >20 to 100 uL use a 100 uL syringe.