Introduction to Spectrophotometer

Lab: Introduction to: spectrophotometery & simulated standard curve of human iron (Fe2+) measurements

Objectives to the lab:

I. To learn how to calibrate the spectrophotometer

II. To practice dilution calculations of glucose.

III.To practice dilution calculation of Fe2+ to obtain a standard curve for Fe2+

IV. To practice graphing a standard curve of Fe2+

V. To determine concentration of a patient’s sample of Fe2+

VI. To interpret the Fe2+ values of your “patients”

VI. To provide you skills to help you perform your upcoming enzyme lab and analyze your data for your enzyme laboratory report

1 2 Supplies: Bring to your bench (1 bench/spectrophotometer)

• Obtain an empty test tube

• Bring approximately 10 ml distilled water in a beaker (simply pour water into beaker)

• Bring 2 transfer pipets to your bench

I. Calibration

 Set wavelength at 580nm

 Adjust Zero Transmission

Note: Your spectrophotometer probably is not digital and you will have to look at the needle and scale for

Wavelength plug

Adjust to Zero Transmission

3  Pipet approximately 5 ml of water into cuvette (about three-fourths full). You simply need adequate amount of water (or sample) to allow light source to encounter sample in its path: this requires between 3-5 ml of fluid.

 Place cuvette into spectrophotometer

Adjust transmission to 100%

4 SPECTROPHOTOMETER

0 100 T A

Set: 100% transmission with cuvette + water Set: 0% transmission without cuvette

5 Practice problems: Part A you will practice calculating a glucose solution. You will then apply this same principle to your dilutions of Fe2+ that you will do on the next page, part B.

PRACTICE PROBLEM:

A) If you had a concentrated “stock” solution of glucose 15 mg/ml and you needed 8 ml of a glucose solution with a concentration of 2 mg/ml, you would set up the problem as follows:

2 mg/ml ------X 8 ml = 1.07 ml 15 mg/ml

You will take 1.07 ml of your 15 mg/ml glucose solution and add 6.93 ml of water

(Note: 8 ml – 1.07 = 6.93)

B) If a problem requires you to perform a “serial” dilution, this means that you must use the previous solution to prepare the next solution.

For example if you made serial dilutions having a final total volume of 8 ml of glucose beginning with the 2 mg/ml for the following dilution:

1.0 mg/ml 0.5 mg/ml 0.25 mg/ml

You would:

1 mg/ml ------X 8 ml = 4 ml of 2 mg/ml + 4.0 ml water 2 mg/ml

Try this one yourself and then you are ready to move onto your own standard curve in part II

0.5 mg/ml ------X 8 ml = ______1 mg/ml

Lastly, in the space below set up the calculation for the serial dilution of 0.25 mg/ml.

What is represents a normal fasting blood glucose level? < 175 mg/dl <150 mg/dl <110 mg/dl

6 II. Standard Curve: Dilutions of Fe2+ & Absorbance Measurements

You are given a solution of known Fe2+ concentration: 10 ug/ml. Your first calculation requires that you determine how much of the 10 ug/ml Fe2+ solution you should dilute to obtain the 2 ug/ml final; then continue with serial dilutions for all of the remaining concentrations. Note you need to make 5 ml volume for each of your dilutions. Arrows mark the columns you must fill in. (note: the absorbance values are given, normally you would have to determine these absorbances after you have diluted the samples.)

Final Tube Fe2+ Water Final Concentration Absorbance at Number (10 ug/ml) (ml) volume (ug/ml) (580 nm) (ml)

1 5 2 1.2

2 5 1 0.65

3 5 0.5 0.35

5 0.25 0.17 4

5 5 0 0

7 IIII. Practice Standard Curve: Name:

Graph your standard curve using the data on the previous page.

Your independent variable is: ______; What is your dependent variable is:______

You place the independent variable on the ______axis and the dependent variable on the: ______axis.

IV. Now: Use your standard curve to determine your unknown patient samples: fill in the concentration of Fe2+ (ug/dl). The column is noted with an arrow.

9 Patient Fe2+ Fe2+ Fe2+ 580 Abs ug/ml dl/ml* Jose 0.26 Paul 0.81 Tiffany 0.70 Gina 1.5 Lester 0.43

These are published examples of the ranges human serum Fe2+ levels.

*Note: dl= deciliter or 1/10th of a liter. A liter= 1000 ml, therefore a dl= 100 ml. Your standard curve is providing per ml values; you must multiply your answers by 100 to obtain dl values.

V. Fe2+ level test

Normal serum Fe2+ values are as follows:

 Adult males: 75-175 micrograms/dL  Adult Fe2+males: 65-165 micrograms/dL  Children: 50-120 micrograms/dL  Newborns: 100-250 micrograms/dL.

DATA INTERPRETATION

A) Which of your “patients” is most likely the “newborn”? ______

B) Which of your patients is most likely a child? ______

C) Which of your patients has abnormally low Fe2+ levels? ______

You will use these principles in your enzyme laboratory.