BIOC - Separation and Identification of Amino Acids Flow Chart PDF

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Summary

Flow Chart for Lab #1...

Description

Experiment 1.1 Clamp Chromatography column over a beaker

Uncap top and bottom of Column allowing fluids to drain

Cap when bottom of fluid meniscus is just on top of resin

Cap till Ready

Wash column allowing 1-2mL of Tris to remain above resin

Pour 5mL of Tris Buffer into measuring cylinder

Obtain 5mL of 1M NaOH

Uncap cylinder allowing buffer meniscus to reach top of resin

Add 5mL of 1M NaOH to column till meniscus touches resin

Micropipette 200μL Amino Acid Mix close to top of resin

Add 5mL of 0.1M Citrate Buffer, Wash column and cap

Cap till Ready

Uncap column tip to allow liquid to level above resin

Discard Beaker contents

Prep 6 test tubes and fill a 7th with water

Add 9mL of 0.1M Tris Buffer to column and fill tubes 4-6 with extra added to 6 (if any)

Collect 3mL of solution in tubes 1-3 with extra added to 3 (if any)

Place column on top of test tube 1 and add 9mL of Citrate to column

Fill reservoir to top with Tris Buffer and let drain

Refill reservoir with Tris Buffer and cap ends ensuring no leaks

Experiment 1.2 Creating Standards Fill new capillary tube with one standard amino acid solution

Holding Microcapillary at 90* angle to TLC plate, touch tip to plate

Let drop dry before next application, total of 3

Standard spot should be small for good res.

Estimate how much standard was spotted

Ensure each spot roughly 2mm in diameter

Apply other 3 standard amino acids to TLC plate

Apply amino acid mixture from 1.1 to TLC plate

Creating Fractions Fill capillary with fractions

Tilt tube and touch surface of liquid with capillary

Apply this method 4 times per fraction, drying each spot with air stream at bench When dry, white-onwhite spots will disappear.

Rest of 1.2 When dry, stand TLC plate, origin down in TLC chamber

Replace lid and allow plate to develop til solvent has gone 2cm down from top

Remove plate from TLC chamber, mark position of solvent front and evaporate solvent in fume hood

Measure distances moved from origin of standards and fraction spots, & calculate Rf

Spots developed through heating TLC plate and indicated on write-up diagram

TA sprays plate to detect separated amino acid spots...