1. GenChem Week 11 Patient Project: Glucose Determination Today’s Agenda:  Review patient project.  Review understanding of absorption spectroscopy.  Develop the day’s problem.  Perform the challenge: determine [glucose] in sample.

2. GenChem Ion-selective electrodes 1415161718 (12) 2122232425 (13) 28293012 Lab Finals Gen. Chem. Exam 4 (14) Looking Ahead Lab Final Exam Building a Poster Last Makeup Lab Final Exam: ??? Gen. Chem. Exam 3 This FRIDAY! Poster Day: Mon, May 5

3. GenChem Instructor & Course Evaluation: In the past we have found our students’ remarks and suggestions to be very helpful. This input has led to a number of changes in our experiments and our programs. For the benefit of future students we ask for your thoughtful comments. But First : What do you think? A.This information will be used not only to evaluate your instructor but also to evaluate the course. B.Your instructor will not be allowed to see your comments until AFTER grades have been submitted. C.ALL evaluations are read by at least three members of the department. D.We need to know what we are doing right as well as how we can improve

4. GenChem We are using the University’s Teacher & Course Evaluation (TCE) Online course evaluation tool. A.You will receive (or may already have) an email from TCE with the URL when the evaluation is available. The site will be closed and data downloaded on Wednesday, April 23 rd. B.You will have about two weeks to respond. The site will be closed and data downloaded on Wednesday, April 23 rd. Instructor & Course Evaluation

5. GenChem The Patient Project  Patient case studies  Patient interview data  Urine analysis (test strips)  CK and LD tests  Glucose test  Ion-selective electrodes  Diagnosis and presentation as a poster

6. GenChem Testing for glucose The question posed to you is “how much glucose is in your samples?” You will use a common clinical test to get an answer. What do you know about these tests:  The test strip  The enzymatic test. What information do they provide?

7. GenChem Glucose Pre-lab

8. GenChem In your groups:  Discuss how you can use Beer’s Law to determine the concentration of something in solution. Given values for  and b, A can be measured and C calculated. Let′s think! Using Spectroscopy to measure concentrations

9. GenChem If we construct a calibration plot from values measured with standards, we can read unknown values directly from the graph. Do we really need ? Consider a sample having an absorbance of 0.37

10. GenChem Creating a Calibration Plot In your groups review your responses to the following pre-lab question and come up with a consensus answer.  Indicate how many standard glucose solutions you expect to make and why you think that will be a sufficient number.  List the glucose concentrations of the standards you expect to make.  Explain why one of the standard solutions needs to contain no glucose. Let′s think! How many total solutions will you need to be measuring the absorbance of?

11. GenChem Patient Sample XX in a 6” test tube Glucose Standard Patient Sample XX 50 mL in a small beaker Solutions in the fume hood Solutions on your bench 10 mL in a 6” test tube Glucose Standard in a small beaker HK GLUCOSE Determination Flowchart (per 4 students) DO THIS FIRST Dilute 25 mL HK reagent with 25 mL DI water (for 50 mL total) Solutions on the reagent bench: HK Reagent Dilute HK Reagent in a small beaker

12. GenChem Patient Sample XX in a 6” test tube Glucose Standard Patient Sample XX 50 mL in a small beaker 2.00 mL “Glucose Standard” + 8.00 mL DI H 2 O 4.00 mL “Glucose Standard” + 6.00 mL DI H 2 O 6.00 mL “Glucose Standard” + 4.00 mL DI H 2 O 8.00 mL “Glucose Standard” + 2.00 mL DI H 2 O Solutions in the fume hood Solutions on your bench 10 mL in a 6” test tube Parallel dilutions Glucose Standard in a small beaker HK GLUCOSE Determination Flowchart (per 4 students) Glucose 2:10 dilution in tube “1” Glucose 4:10 dilution in tube “2” Glucose 6:10 dilution in tube “3” Glucose 8:10 dilution in tube “4” DO THIS FIRST DO THIS SECOND Dilute 25 mL HK reagent with 25 mL DI water (for 50 mL total) Solutions on the reagent bench: HK Reagent Dilute HK Reagent in a small beaker

13. GenChem Patient Sample XX in a 6” test tube Glucose Standard Patient Sample XX 50 mL in a small beaker 2.00 mL “Glucose Standard” + 8.00 mL DI H 2 O 4.00 mL “Glucose Standard” + 6.00 mL DI H 2 O 6.00 mL “Glucose Standard” + 4.00 mL DI H 2 O 8.00 mL “Glucose Standard” + 2.00 mL DI H 2 O Test Tube “0 ” Test Tube “1 ” Test Tube “2 ” Test Tube “3 ” Test Tube “4 ” Test Tube “5 “ Test Tube “XX” 4.00 mL Dilute HK Reagent + 1.00 mL “1” 4.00 mL Dilute HK Reagent + 1.00 mL “Glucose Standard” 4.00 mL Dilute HK Reagent + 1.00 mL “Patient Sample XX” 4.00 mL Dilute HK Reagent + 1.00 mL DI H 2 O Solutions in the fume hood Solutions on your bench 10 mL in a 6” test tube Parallel dilutions Glucose Standard in a small beaker HK GLUCOSE Determination Flowchart (per 4 students) Glucose 2:10 dilution in tube “1” Glucose 4:10 dilution in tube “2” Glucose 6:10 dilution in tube “3” Glucose 8:10 dilution in tube “4” DO THIS FIRST DO THIS SECOND 4.00 mL Dilute HK Reagent + 1.00 mL “2” 4.00 mL Dilute HK Reagent + 1.00 mL “3” 4.00 mL Dilute HK Reagent + 1.00 mL “4” Dilute 25 mL HK reagent with 25 mL DI water (for 50 mL total) DO THIS THIRD Solutions on the reagent bench: HK Reagent Dilute HK Reagent in a small beaker Dilutions with HK added

14. GenChem Patient Sample XX in a 6” test tube Glucose Standard Patient Sample XX 50 mL in a small beaker 2.00 mL “Glucose Standard” + 8.00 mL DI H 2 O 4.00 mL “Glucose Standard” + 6.00 mL DI H 2 O 6.00 mL “Glucose Standard” + 4.00 mL DI H 2 O 8.00 mL “Glucose Standard” + 2.00 mL DI H 2 O Test Tube “0 ” Test Tube “1 ” Test Tube “2 ” Test Tube “3 ” Test Tube “4 ” Test Tube “5 “ Test Tube “XX” 4.00 mL Dilute HK Reagent + 1.00 mL “1” 4.00 mL Dilute HK Reagent + 1.00 mL “Glucose Standard” 4.00 mL Dilute HK Reagent + 1.00 mL “Patient Sample XX” 4.00 mL Dilute HK Reagent + 1.00 mL DI H 2 O Solutions in the fume hood Solutions on your bench 10 mL in a 6” test tube Parallel dilutions Glucose Standard in a small beaker HK GLUCOSE Determination Flowchart (per 4 students) Glucose 2:10 dilution in tube “1” Glucose 4:10 dilution in tube “2” Glucose 6:10 dilution in tube “3” Glucose 8:10 dilution in tube “4” DO THIS FIRST DO THIS SECOND 4.00 mL Dilute HK Reagent + 1.00 mL “2” 4.00 mL Dilute HK Reagent + 1.00 mL “3” 4.00 mL Dilute HK Reagent + 1.00 mL “4” Dilute 25 mL HK reagent with 25 mL DI water (for 50 mL total) DO THIS THIRD Solutions on the reagent bench: HK Reagent Dilute HK Reagent in a small beaker IMPORTANT! Configure (set-up) spectrometer to read at 340 nm before taking any readings! Use as the “blank” in calibrating the spectrometer Read the absorbance of dilution in tube “1” DO THIS FORTH Read the absorbance of dilution in tube “2” Read the absorbance of dilution in tube “3” Read the absorbance of dilution in tube “4” Read the absorbance of “5” Read the absorbance of “5” Absorbance readings with Logger Pro Read the absorbance of “XX” IMPORTANT! Patient Sample “XX” must be read at the same time as the calibration dilutions! Dilutions with HK added

15. GenChem

16. Creating a Calibration Plot In your groups review your responses to the following pre-lab question and come up with a consensus answer. Write a detailed procedure for how you expect to go about preparing your standard and sample solutions. This procedure must include answers to the following questions  how much of each solution should be made?  what glassware should be used to measure the volumes? Let′s think!

17. GenChem Divvying up tasks Decide in your group who is going to do what.

18. GenChem Performing the reactions safely. You are required to wear gloves when performing this experiment.

19. GenChem Performing the Test Reaction Reminders Syringes 1)Syringes are provided for this (note that this is a change from the guide). 2)Use DI water 3)DO NOT shake the test tubes (this could damage the enzymes in the reagent). Leave the parafilm® in place to avoid evaporation. 4)Once the reagents have been mixed, allow the reaction to proceed undisturbed for 10 minutes at room temperature. Let′s think!

20. GenChem Yet one more waste stream. Syringe needles are sharps waste and need to be disposed of accordingly.

21. GenChem Your Challenge Prepare your standards Prepare your samples Perform the reactions Measure their absorbances. Available resources:  Standard glucose solution.  HK reagent.  automatic pipet and various plastic syringes.  various glassware.  Spectrometer. Design and implement an experimental procedure! Let′s explore! You have 90 minutes

22. GenChem Let′s think! Data Evaluation  Inspect your absorbance values. Are they suitable? Should any be discarded?  Plot your calibration data.  Inspect your calibration plot. Is it suitable? Are more data points required?  Inspect your sample value. Is it within the range of your standard values?  Do you have the data you need or are additional measurements required? Make a list of additional solutions to be prepared and measurements to be made

23. GenChem Followup Challenge Prepare additional standards and samples as required. Measure their absorbance Available resources:  Standard glucose solution.  HK reagent.  automatic pipet and various plastic syringes.  various glassware.  Spectrometer. Design and implement an experimental procedure! Let′s explore! You have 30 minutes

24. GenChemWrap-up Determine the [glucose] in your patient samples. progress report You will report your results in a progress report constructed next week in class.