Why is it important to thoroughly vortex the contents of the vial after adding reagent 4 (Steps 3 and 4., stage 1). To ensure that the two immiscible solvents come into contact with each other, permitting the derivatization reactions to occur. B.To ensure that the solution is mixed thoroughly. To ensure that all the amino acids are transferred to the lower aqueous layer so that a full extraction occurs. To remove all the interferring compounds by transferring them to the upper organic layer Hint:Reagent 4 is the derivatizing agent dissolved in octane. 2. Store the prepared eluting medium at room temperature. At the end of theexercise, discard any leftover eluting medium.Step 1.Addition of internal standard to the sample1. For each sample, line up one sorbent tip and one glass sample preparationvial in the vial rack. Match vials and sorbent tips before proceeding to ensurethat the sorbent tip will reach the bottom of the sample preparation vial.2. Pipette 100 μl of Reagent 1 (Internal Standard Solution) and 100 µl of sampleinto each sample preparation vial.Step 2.Solid phase extraction1. Attach a sorbent tip to a 1.5 ml syringe and loosen the syringe piston;immerse the tip and let the solution in the sample preparation vial passthrough the sorbent tip by VERY SLOWLY pulling back the syringe piston, inSMALL steps.NOTES:●●●The sorbent tip should stay in the sample preparation vial, even whiledispensing reagents in the later steps. If the sorbent tip cannot reach thebottom of the vial, tilt the vial to about 45°, push the tip into the vial gently andproceed.As with any solid phase extraction procedure, it is vital to load the sampleslowly on to the solid sorbent. To ensure this, watch as the liquid accumulatesinside the syringe barrel and move the piston only as the accumulation slowsdown.If you run out of piston range, detach the sorbent tip, expel the solution fromthe syringe barrel, then reattach the sorbent tip and proceed.2. Pipette 200 μl Reagent 2 (Washing Solution) into the same samplepreparation vial.3. Pass the solution VERY SLOWLY through the sorbent tip and into the syringebarrel. Drain the liquid from the sorbent bed by pulling air through the sorbenttip.4. Detach the sorbent tip, and leave it in the sample preparation vial, thendiscard the liquid accumulated in the syringe.Do not dispose of the 1.5 ml syringes as they can be washed and reused withother samples.5. Pipette 200 μl eluting medium (prepared earlier) into the same samplepreparation vial.6. Pull back the piston of a 0.6 ml syringe halfway up the barrel and attach thesorbent tip used above. 7. Wet the sorbent with the eluting medium by slowly pulling back the syringepiston; watch as the liquid rises through the sorbent particles and stopapproximately when the liquid reaches the filter plug in the sorbent tip.Take care to ensure that the liquid does not pass through the sorbent tip andinto the syringe as this will contaminate the syringe.8. Eject the liquid and the sorbent particles out of the tip and into the samplepreparation vial.9. Repeat steps 7 to 9 until all sorbent particles in the tip are expelled into thesample preparation vial. Only the filter disk should remain in the empty tip.Discard the empty tip in a suitable waste container. Again keep the syringe asit can be cleaned and reused with other samples.Steps 3 and 4. Derivatization and Liquid-Liquid Extraction1. Use the adjustable Drummond Microdispenser to transfer 50 µl Reagent 4into the sample preparation vial.NOTES:OYour instructor will demonstrate how to use the Microdispensercorrectly.O Avoid cross-contamination by not touching the inner wall of the samplevial with the tip of the Microdispenser. The piston will ensure propertransfer of liquids into the vial without the need of touching the vial wall.Use the same Microdispenser with both Reagents 4 and 5. It is notnecessary to change Microdispenser tips between uses, or to wash thedispenser between uses of Reagent 4 and Reagent 5.O2. Emulsify the liquid in the vial by repeatedly vortexing it for around 5-8seconds. During vortexing hold the sample vial firm so that no liquid comesout of the vial. Afterwards leave the sample for 1 minute for the reactions toproceed.3. Re-emulsify the liquids in the vial by vortexing again for about 5 seconds.Again allow the reaction to proceed for another minute after vortexing.4. Transfer with the Microdispenser 100 µl Reagent 5 (2 x 50 µl forconvenience) into the sample vial and mix for about 5 seconds. Again leavethe reaction to proceed for one more minute.5. Transfer part of the (upper) organic layer (about 50 µl) into an autosamplervial using a Pasteur pipette. It is critical to avoid transfer of the (bottom)aqueous layer along with the organic layer, even if it means that some of theorganic layer is left behind6. Evaporate the contents of the autosampler vial to (almost) dryness in aGENTLE stream of nitrogen. Do not leave the samples in the nitrogen streamfor more than 10 minutes.

A vortex mixer or vortexer is a basic device used in laboratories to mix small vials of liquid. It…

Why is it important to thoroughly vortex the contents of the vial after adding reagent 4 (Steps 3 and 4., stage 1). To ensure that the two immiscible solvents come into contact with each other, permitting the derivatization reactions to occur. B.To ensure that the solution is mixed thoroughly. To ensure that all the amino acids are transferred to the lower aqueous layer so that a full extraction occurs.

Why is it important to thoroughly vortex the contents of the vial after adding reagent 4 (Steps 3 and 4., stage 1). To ensure that the two immiscible solvents come into contact with each other, permitting the derivatization reactions to occur. B.To ensure that the solution is mixed thoroughly. To ensure that all the amino acids are transferred to the lower aqueous layer so that a full extraction occurs.

EBK A SMALL SCALE APPROACH TO ORGANIC L

4th Edition

ISBN:9781305446021

Author:Lampman

Publisher:Lampman

Chapter20: Nucleophilic Substitution Reactions: Competing Nucleophiles

Section: Chapter Questions

Problem 8Q

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