DEPARTMENT OF LIFE SCIENCES

BIOC 2360
PRACTICAL # 1

Instrumentation in the Laboratory

Student Learning Outcomes

At the end of this lab, you should be able to:

 Use the pH meter to measure pH and neutralize solutions.

 Use the micropipettor.
 Use the spectrophotometer.
 Construct and utilize a calibration curve and perform associated calculations.

MEASURING pH

The pH meter is a simple machine used to measure pH. It has two main components - an
electrode which is usually on a stand and the base immersed in buffer or distilled water and a
dashboard which has a display.

Measuring the pH of a Sample

1. Always ensure that the sample is at room temperature and well stirred.

2. Remove the electrode from the storage solution and rinse with distilled water. Shake gently
to remove excess water.

3. Pour the solution into a large test tube and immerse the electrode into sample swirling (if
possible) while you wait for the reading. Some pH meters will say “ready” at which point
you record the reading displayed. Other pH meters you will have to wait for the reading to
stabilize. Once the reading is stable for 5-7 seconds, record it.

4. The electrode must be thoroughly rinsed with distilled water before proceeding to the next
sample.

5. When finished reading samples, always rinse the electrode, replace on the stand and ensure
that the base of the electrode is well immersed in distilled water/buffer solution.

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BIOC2069
Lab 1 – Instrumentation
Semester I 2014/2015
EXERCISE 1: Determination of pH of Various Solutions

Using the method above, measure and record the pH of 7mL of the following solutions:

a) Coca cola
b) 0.25M NaOH (save this for Exercise 2)
c) 10% Acetic acid

EXERCISE 2: Adjusting the pH of a Solution

Adjusting the pH of a solution to a specific pH is a tricky task as small additions of excess acid
or base can easily cause the pH to dramatically increase as you approach neutralization. The best
way to do this is by using various decreasing concentrations of the titrant and monitoring the pH
along the procedure.

Procedure

You will be provided with 0.5M HCl, 0.25 M HCl and 0.1M HCl solutions on the centre bench.

1. Place the electrode into your sample (b) (0.25M NaOH) from above.

2. Using a Pasteur pipette in a drop wise manner directly introduce 0.5M HCl until the pH nears
6.8-7.2. Ensure that after each addition of acid, the solution is well vortexed or swirled before
taking the final pH reading. It is not necessary to rinse or remove the electrode from the
solution during the procedure.

3. Once the pH is around 6.8-7.2, use the 0.25M HCl to adjust the pH to around 6.

4. Use the 0.1 M HCl to further adjust the pH to 5.

5. Adjust the volume to 10mL with distilled water. If you pass the 10mL mark, measure and
record the final volume.

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BIOC2069
Lab 1 – Instrumentation
Semester I 2014/2015
Exercise 3: Using the micropipette

a) Small-Volume Micropipettor
This exercise simulates setting up a reaction, using a micropipettor with a range of 1-20 µL.

1. Use a permanent marker to label three 1.5-mL tubes A, B, and C.

2. Use the matrix below as a checklist while adding solutions to each reaction tube.

Tube number Solution 1 Solution 2 Solution 3 Solution 4 Total volume

A 4 µL 4 µL 2 µL -

B 4 µL 4 µL - 2 µL

C 3 µL 3 µL 2 µL 2 µL

3. Set the micropipettor to 4 µL and add Solution 1 to each reaction tube.

4. Use a fresh tip to add appropriate volume of Solution 2 to a clean spot on reaction Tubes A, B,
and C.

5. Use a fresh tip to add 2 µL of Solution 3 to Tubes A and C.

6. Use a fresh tip to add 2 µL of Solution 4 to Tubes B and C.

7. Close tops. Pool and mix reagents by using one of the following methods:

a. Sharply tap the tube bottom on the bench top. Make sure that the drops have pooled into one
drop at the bottom of the tube.

or

b. Place the tubes in a microfuge and apply a short few-second pulse. Make sure that the reaction
tubes are placed in a balanced configuration in the microfuge rotor. Spinning tubes in an
unbalanced position will damage the microfuge motor.

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BIOC2069
Lab 1 – Instrumentation
Semester I 2014/2015
8. A total of 10 µL of reagents was added to each reaction tube. To check the measurements were
accurate, set the pipette to 10 µL and very carefully withdraw solution from each tube.

a. Is the tip just filled? or

b. Is a small volume of fluid left in tube? or

c. After extracting all fluid, is an air space left in the tip end? (The air can be displaced and actual
volume determined simply by rotating, volume adjustment to push fluid to very end of tip. Then,
read the volume directly).

b) Large-Volume Micropipettor
This exercise simulates a bacterial transformation or plasmid preparation, for which a 100-1000
µL micropipettor is used. It is far easier to mismeasure when using a large-volume micropipettor.
If the plunger is not released slowly, an air bubble may form or solution may be drawn into
piston.

1. Use a permanent marker to label two 1.5-mL reaction tubes D and E.

2. Use the matrix below as a checklist while adding solutions to each reaction tube.

Make a note in your book of which pipetman you used for each volume pipetted.

Tube number Solution 1 Solution 2 Solution 3 Solution 4 Total volume

D 100 µL 200 µL 150 µL 550 µL

E 150 µL 250 µL 350 µL 250 µL

3. Set the micropipettor to add appropriate volumes of Solutions 1-4 to reaction tubes D and E.
Follow the same procedure as for the small-volume micropipettor exercise.

4. A total of 1000 µL of reactants was added to each tube. To check that measurements were
accurate, set the micropipettor to 1000 µL and carefully withdraw solution from each tube.

5. a. Is the tip just filled? or

b. Is a small volume of fluid left in tube? or

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BIOC2069
Lab 1 – Instrumentation
Semester I 2014/2015
c. After extracting all fluid, is an air space left in the tip end? (The air can be displaced and actual
volume determined simply by rotating volume adjustment to push fluid to very end of tip. Then,
read the volume directly.)

SPECTROPHOTOMETRY & CALIBRATION CURVES

To construct a calibration curve, you must be provided with a standard solution of known
concentration. Because in biochemistry many compounds which we wish to quantify is usually
clear, it is common that other reagents are added to produce a coloured compound. To make use
of a calibration curve, you set up a series of tubes varying amounts of substance of known
concentration in them. It is best practice to add the compounds which result in the development
of the chromophore at the same time that you prepare your calibration curve. One of the tubes
will contain none of the reagent being quantified (this is called your zero or blank tube). This is
used to blank the spectrometer and serves to remove any background that may be due to other
compounds that may absorb. After blanking the spectrophotometer, you can proceed to measure
the absorbance of your remaining tubes.

Measuring Samples

1. To measure the sample, a small plastic (or glass) container is provided called a cuvette. To
ensure an accurate reading is acquired, ensure there are no scratches on the clear sides of the
cuvette.

2. Turn on the spectrophotometer (switch to the back) at least 5 minutes before taking your first
reading. While it is warming up, using the nm button, set your desired absorbance.

3. Pour the sample containing the blank into a clean cuvette so that it is a little more than half
filled. Ensure that the sample is at room temperature and there are no bubbles in the cuvette.

4. Open the lid and you will see a small arch or perforation (this is where the beam of light will
be emitted). Place the cuvette firmly in the holder so that the clear side faces this arch. Cover
and press “zero” or “blank”. The value which appears should be 0.000.

5. Remove sample, pour sample back into the test tube from which it was taken, rinse with
water and place your new sample into the cuvette.

6. For this and subsequent samples, place the cuvette in the holder, cover and record the
reading.

7. Rinse between each sample.

Plot the curve (remember it is actually a straight line through the origin) of absorbance at the
particular wavelenght (y-axis) vs. amount of compound (mg, g, micromoles etc on the x-axis).
You then draw your best-fit straight line through the origin. (Why must this line pass
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BIOC2069
Lab 1 – Instrumentation
Semester I 2014/2015
through the origin??) The absorbance value of the unknown is read on the spectrophotometer and
should fall within the line of your curve. Once you have determined the amount of your
unknown you can work back to determine its concentration. For your lab submissions, all
calibration curves must be digitally drawn with the equation written on the same page next to the
line (use Excel). The equation can be used to determine the amounts in your unknowns.
MATERIALS

Reagent A: 2%Na2CO3 on 0.1M NaOH

Reagent B: 0.5% CuSO4.5H2O in 1% sodium citrate

Reagent C: Mix just before using. Combine Reagent A and Reagent B in a 50:1 mL ratio

Diluted Folin Cicocalteau’s Reagent (1:1 with water) this has been provided with you

Exercise 4: Protein Determination by the Lowry Method

Preparation of the Protein Calibration curve

1. Prepare a calibration curve by pipetting 0, 0.1, 0.2, 0.3, 0.5, 0.8 and 1.0 mL of the
provided standard protein solution (BSA, 200 µg/mL). Make the volumes up to 1.0 mL
with distilled water.

2. Add 5 mL of (Lowry) Reagent C and leave standing for EXACTLY 10 minutes

3. Add 0.5 mL of diluted Folin-Cocicalteau’s solution, mix well and leave the tubes
standing for 30 minutes at room temperature.

4. Read the absorbance against the blank (tube 1) at 750nm. Plot a calibration curve of
absorbance against milligrams of protein.

Preparation of the Protein Calibration curve

1. Pipette 0.1, 0.2 and 0.5 mL of your unknown samples into a clean labeled test tubes.
Bring the volumes in the tubes up to 1.0 mL with distilled water and proceed with the
Lowry Assay.

Plot a calibration graph relating absorbance at 750nm to BSA concentration (μg/mL). Using this
calibration curve, determine the amount of protein found in the unknown samples.
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BIOC2069
Lab 1 – Instrumentation
Semester I 2014/2015