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.
Chemistry
10th Edition
ISBN:9781305957404
Author:Steven S. Zumdahl, Susan A. Zumdahl, Donald J. DeCoste
Publisher:Steven S. Zumdahl, Susan A. Zumdahl, Donald J. DeCoste
Chapter1: Chemical Foundations
Section: Chapter Questions
Problem 1RQ: Define and explain the differences between the following terms. a. law and theory b. theory and...
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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.

Transcribed Image Text:2. Store the prepared eluting medium at room temperature. At the end of the
exercise, discard any leftover eluting medium.
Step 1.
Addition of internal standard to the sample
1. For each sample, line up one sorbent tip and one glass sample preparation
vial in the vial rack. Match vials and sorbent tips before proceeding to ensure
that 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 sample
into each sample preparation vial.
Step 2.
Solid phase extraction
1. 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 pass
through the sorbent tip by VERY SLOWLY pulling back the syringe piston, in
SMALL steps.
NOTES:
●
●
●
The sorbent tip should stay in the sample preparation vial, even while
dispensing reagents in the later steps. If the sorbent tip cannot reach the
bottom of the vial, tilt the vial to about 45°, push the tip into the vial gently and
proceed.
As with any solid phase extraction procedure, it is vital to load the sample
slowly on to the solid sorbent. To ensure this, watch as the liquid accumulates
inside the syringe barrel and move the piston only as the accumulation slows
down.
If you run out of piston range, detach the sorbent tip, expel the solution from
the syringe barrel, then reattach the sorbent tip and proceed.
2. Pipette 200 μl Reagent 2 (Washing Solution) into the same sample
preparation vial.
3. Pass the solution VERY SLOWLY through the sorbent tip and into the syringe
barrel. Drain the liquid from the sorbent bed by pulling air through the sorbent
tip.
4. Detach the sorbent tip, and leave it in the sample preparation vial, then
discard the liquid accumulated in the syringe.
Do not dispose of the 1.5 ml syringes as they can be washed and reused with
other samples.
5. Pipette 200 μl eluting medium (prepared earlier) into the same sample
preparation vial.
6. Pull back the piston of a 0.6 ml syringe halfway up the barrel and attach the
sorbent tip used above.

Transcribed Image Text:7. Wet the sorbent with the eluting medium by slowly pulling back the syringe
piston; watch as the liquid rises through the sorbent particles and stop
approximately 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 and
into the syringe as this will contaminate the syringe.
8. Eject the liquid and the sorbent particles out of the tip and into the sample
preparation vial.
9. Repeat steps 7 to 9 until all sorbent particles in the tip are expelled into the
sample 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 as
it can be cleaned and reused with other samples.
Steps 3 and 4. Derivatization and Liquid-Liquid Extraction
1. Use the adjustable Drummond Microdispenser to transfer 50 µl Reagent 4
into the sample preparation vial.
NOTES:
O
Your instructor will demonstrate how to use the Microdispenser
correctly.
O Avoid cross-contamination by not touching the inner wall of the sample
vial with the tip of the Microdispenser. The piston will ensure proper
transfer 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 not
necessary to change Microdispenser tips between uses, or to wash the
dispenser between uses of Reagent 4 and Reagent 5.
O
2. Emulsify the liquid in the vial by repeatedly vortexing it for around 5-8
seconds. During vortexing hold the sample vial firm so that no liquid comes
out of the vial. Afterwards leave the sample for 1 minute for the reactions to
proceed.
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 for
convenience) into the sample vial and mix for about 5 seconds. Again leave
the reaction to proceed for one more minute.
5. Transfer part of the (upper) organic layer (about 50 µl) into an autosampler
vial 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 the
organic layer is left behind
6. Evaporate the contents of the autosampler vial to (almost) dryness in a
GENTLE stream of nitrogen. Do not leave the samples in the nitrogen stream
for more than 10 minutes.
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