Tearing my hair out with amylase starch test experiment.

[bodil]

Hi all,

 

Am trialing an amylase starch experiment with variable pH for the students and it is causing major angst.

 

TRIAL 1.

 

BUFFERS - I made buffers of pH 3-11 using the CLEAPSS recipes (http://mityeast.pbworks.com/w/file/fetch/83078368/http---www.cleapss.org.uk-attachments-article-0-RBPrint.pdf) page 21. I tested the buffers using a 2-point calibrated probe with standardised buffers 4 and 10. All buffers were within .2 of the desired value.

 

AMYLASE - Used a laboratory sourced supply which is apparently CLARASE G fungal alpha amylase. Made 9 x 1.0% solutions using each buffer.

 

STARCH - Used laboratory grade soluble starch for idiometry. Made 9 x 0.5% solutions for each pH.

 

INDICATOR - Made a 0.1M KI3 solution with distilled water.

 

METHOD - Pipetted 4ml of each starch soln into a vial and added 100 microlitres of KI3 indicator. Checked for uniform colour in all vials. Pipetted 1.0ml of diastase into each vial. Started timer and recorded time for colour to disappear,

 

RESULTS - pH 3-4 did not change colour after 30 minutes. Evidence of cloudiness in amylase samples suggested amylase denatured upon addition to buffer. pH 5-8 took decreasing times to decolourise. pH 9-11 decolourised instantaneously upon addition of amylase. All samples repeated 3 times.

 

CONCLUSION - Optimal pH in the 9-11 range. Not happy!

 

TRIAL 2.

 

Used same buffers and soluble starch in buffer solutions. Changed amylase to 1.0% brewing alpha amylase in distilled water. Repeated trials.

 

CONCLUSION - Optimal pH in the 9-11 range. Not happy at all!

 

TRIAL 3.

 

Used same buffers and laboratory grade amylase but changed starch to 1% corn starch using paste in boiling deionised water. Allowed to cool.

 

METHOD - Pipetted 2ml of starch soln into a vial and added 2.0ml of appropriate buffer. Added 100 microlitres of KI3 indicator. Checked for uniform colour in all vials. Pipetted 1.0ml of diastase into each vial. Started timer and recorded time for colour to disappear.

 

CONCLUSION - Optimal pH in the 9-11 range. Getting angry now!

 

TRIAL 4

 

In exasperation I used a solution of my own saliva in deionised water (I know I wouldn't get the students to do that!!!). Used same method as in trial 3.

 

CONCLUSION - Optimal pH in the 9-11 range. I even tested the pH of the solutions after the trial and got 3.11, 4.15, 5.22, 6.22, 7.02, 7.84, 8.97, 9.69 and 10.30 so pH had not changed massively in terms of the entire pH range and results still way off reported 6.7-7.0 for salivary amylase. Just plain right stumped now (and still angry)!

 

Any help appreciated. If I can't get it to work what chance have the students!! Thanks