SAFETY PRECAUTIONS INSIDE THE LABORATORY

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RULES ON LABORATORY SAFETY
1. During an experiment, place your bag, books, and other materials on the bag rack. Do
not bring unnecessary items to the working area.

2. Switch your cell phone to silent mode during class hours.

3. Do not eat or drink inside the laboratory.

4. Study the experiment before coming to class. Familiarize yourself with the precautions
and the reagents to be observed and used.

5. Know where the safety equipment (e.g., first-aid kit, fire extinguisher, etc.) is located.

6. Report all accidents (even minor ones) or accident-prone situations to the laboratory
instructor.

7. Do not perform any unauthorized experiments. Aside from posing a risk to yourself
and to others, you might also waste time and resources.

8. Assume all chemicals are hazardous. Handle reagent bottles carefully when
transferring or pouring chemicals. Return these containers to their designated cabinets
and place them at a safe distance from table edges.

9. Most reagents are flammable and volatile. Always keep their containers' lids closed,
especially after use. Keep them away from open flames and other sources of ignition.

10. Some organic chemicals are irritating, corrosive, toxic, or poisonous. Handle them
with care and observe the necessary precautions. Wear gloves when using such
chemicals and wash your hands thoroughly afterwards.

11. Read labels carefully before using any reagent. Dispense only the amount needed to
avoid wastage and leave some for the other classes or groups. Do not use unlabeled
reagents.

12. Do not pour excess chemicals back to the reagent bottles. Doing so may contaminate
the stock, especially if a chemical is mistakenly placed in the wrong bottle. Dispose of
the waste and the excess reagents in the designated bins. Do not pour them down the
sink.

13. Use a new pipette for each reagent. Use only a rubber aspirator to pipet chemicals.

14. Never taste or smell the chemicals.

15. Do not clean spillage with a piece of cloth, especially if the chemical is corrosive.
Inform the laboratory instructor of the incident immediately so that proper measures can
be taken.

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16. Dispose of solid wastes (e.g., solid chemicals, piece of scratch paper, etc.) in the
trash can. Do not throw them over the sink to avoid clogging.

17. Laboratory glassware must be washed with a detergent after use to keep them clean
and dry for the next group. Hot glassware should be allowed to cool to room temperature
before being washed to prevent breakage due to thermal shock.

18. In case of glass breakage, sweep the pieces with a broom and collect them with a
dust pan, do not pick them up with your bare hands. Do not use chipped or cracked
glassware especially when heating.

19. Do not put sharp objects like pipettes or droppers inside the pockets of your
laboratory gown.

20. When plugging a laboratory instrument (e.g., a hot plate), check the outlet voltage
first.

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Instrumentation

pH METER
A pH meter uses an electrode to measure the hydrogen ion concentration and the pH of
an unknown solution. The term pH is used to designate the intensity or degree of acidity.
The value of pH, the logarithm of the reciprocal of the hydrogen ion concentration in the
solution, is determined by measuring the difference in the potential of two electrodes
immersed in a sample solution. The pH meter has many applications in medicine,
chemistry, biology, etc.

A typical pH meter is equipped with a glass electrode and a reference electrode or a

single probe combination electrode connected to an electronic meter that measures and
displays the pH reading. Single probe combination electrodes contain the reference and
glass electrodes inside them as shown in Figure 1.

The glass electrode consists of a thin, pH-sensitive glass membrane, the inside of which
contains a solution with a constant concentration of hydrogen ions. When the electrode is
immersed in an unknown solution, the thin glass membrane interacts with the small
hydrogen ions in the solution. The concentration outside the glass membrane depends on
the activity of the hydrogen ions in the solution. The concentration difference results in
the potential across the glass membrane which is measured by a potentiometer.

The most commonly used reference electrode is the calomel electrode which
incorporates a salt bridge filled with a saturated potassium chloride (KCI) solution. The
reference electrode does not change its voltage but sets a standard voltage level to which
the glass electrode is compared.

Most modern pH meters also have a temperature probe which allows for automatic
temperature corrections since pH may vary relative to the temperature.

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STANDARD OPERATING PROCEDURES IN USINGA pH METER
Calibration

To give an accurate pH reading, a pH meter must be calibrated prior to use.

 If the pH of the solution to be tested falls between 7.0 to 10, use the pH 7 and pH
10 calibration buffers for the two-point calibration.
 If the pH of the solution to be tested falls between 4.0 to 7.0, use the pH 4 and
pH 7 calibration buffers for the two-point calibration.

Procedure:

1. Rinse the probe tip with distilled water and blot dry with a tissue paper.

2. Place the tip of the electrode in the first buffer solution. Press "Cal”.

3. Take the pH reading once the reading becomes stable.

4. Rinse the electrode tip with distilled water and blot dry with tissue paper.

5. Immerse the electrode in the second buffer solution. Press "Cal."

6. Take the pH reading once the pH reading becomes stable.

7. Rinse the electrode tip and blot-dry with tissue paper.

Note: The pH readings obtained should correspond with the pH used in the calibration.

pH Measurement of a Sample

1. Adjust the temperature of the sample to room temperature.

2. Rinse the electrode with distilled water and blot-dry with tissue paper.

3. Immerse the electrode in the sample solution. Allow the meter to stabilize then take
the pH reading.

4. Rinse the electrode with distilled water and blot-dry with tissue paper.

5. Return the electrode to its filing solution.

Do's and Don'ts in Using a pH Meter

 Do calibrate the pH probe frequently to ensure accurate results.

 Do rinse the pH probe thoroughly with distilled or deionized water before
measuring samples.
 Do store the pH probe sensing tip in a soaking solution and never in plain water.
The soaking solution may be a pH 7 or a pH 4 buffer solution or a potassium
chloride (KCl) solution recommended by the manufacturer.
 Do not touch the pH probe sensing tip with your bare hands.

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  Do not scratch or damage the pH bulb. If scratched or damaged, the pH electrode
may produce erroneous results.

SPECTROPHOTOMETRY

Colorimetric assays are used to measure the amount of light transmitted through a
sample at a given wavelength. The amount of light absorbed by a substance may be
measured by instruments called spectrophotometers. A spectrophotometer that makes the
sample absorb light in the visible and near ultraviolet region of the electromagnetic
spectrum is called an ultraviolet-visible (UV-Vis) spectrophotometer.

UV-Vis Spectrophotometry

The essential parts of a UV-Vis spectrophotometer consist of a white light source, a

monochromator to disperse light into its component wavelengths, a sample compartment
that holds the transparent tube or cell containing the sample, a detector to measure the
amount of light transmitted, and a read-out device to display the reading. A tungsten
lamp is used as a light source in the visible region while a deuterium lamp is used in the
ultraviolet region.

Light absorbance is based on the capacity of a solution to absorb radiant energy. If a

radiant energy of intensity 10 is allowed to pass through a solution placed in a
transparent tube, the solution absorbs a portion of this radiant energy. The unabsorbed
radiant energy is transmitted. If the intensity of the transmitted energy is 1, the
transmittance T is ho. Although a spectrophotometer detects the transmittance, it is the
absorbance that has a linear relationship with the concentration of the absorbing species
in the analyte solution.

The relationship between transmittance and absorbance is shown in the following

equation:

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The determination of the concentration of the absorbing species in the analyte solution is
based on the Beer-Lambert's Law. According to this law, absorbance is directly related
to the concentration if the measurements are made at a fixed wavelength in a cell of
constant path which is usually 1 em. The law may be expressed by the by the equation:

The molar extinction coefficient is a measure of how strongly a solution absorbs light in
a particular wavelength. If the molar extinction coefficient is known, then the
concentration of a solution can be determined from the absorbance. For two solutions of
the same compound, one with a known concentration (standard solution) and the other
with an unknown concentration, the concentration of the unknown solution can be
calculated from the measured absorbance of the unknown solution and the absorbance of
the standard solution determined under identical conditions using this equation:

When a large number of samples are being run, it is preferable to construct a calibration
or standard curve. Standard curves may be obtained by measure absorbance of a series of
standard solutions whose concentration is in the range of the solution being measured.
Each standard solution must be prepared in exactly the way as the solution being
measured. The absorbance values of the standard solutions are then plotted against the
concentration and the concentration of the solution is read directly from the graph.

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PREPARATION OF STANDARD
THE CALIBRATION CURVE

Graphical Method

1. Comnpute the concentration of the stock reagent made in the experiment.

For example, an albumin stock solution is prepared by dissolving 100 mg albumin in

enough distilled water to make a 100 mL-solution. The concentration of the stock
solution may be computed as follows:

2. Make the proper dilution of the stock solution as described in the procedure and
compute the concentration of the standard solutions using the dilution formula.

Note: The first test tube is usually the reagent blank or distilled water.

For example, in the second test tube, 0.1 mL of the albumin stock solution is pipetted
out, combined with 2.2 mL of the coloring agent, and diluted with 7.7 mL of the distilled
water. The final volume of the solution is 10.0 mL and the concentration of this solution
using the dilution formula is:

The concentration of the standard solutions in the succeeding test tubes must be
calculated in the same manner.

3. Read the absorbance of the standard solutions and the sample solution against the
blank reagent using a spectrophotometer at the specified wavelength.

4. Using a graphing or cross-section paper, plot the absorbance (y-axis) and the
concentration of the standard solutions (-axis). Label the graph properly, including the
units of measurement used and the specified wavelength as shown below.

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5. Determine the "best straight" line among the set of x and y data pairs.

6. Determine the concentration of the sample solution directly from the graph. For
example, if the absorbance of the sample solution is 0.4, then its concentration from the
graph is estimated to be 0.062 mg/mL.

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Using Microsoft Office Excel
A spreadsheet like Microsoft Office Excel may also be used to generate a standard
calibration curve.

1. Open the Excel program by clicking the START button at the lower left corner of the
screen. Point the mouse to ALL PROGRAMS and then select MICROSOFT OFFICE
EXCEL. An empty spreadsheet will appear on the screen.

2. Under column A, enter the concentration of the standards (mg/mL) from the assay
experiment.

3. Under column B, enter the absorbance values obtained from the standard solutions.

4. Once entered, highlight the data by holding the moouse button and dragging the arrow
to form a box around the numbers.

5. From the menu heading, click INSERT, and select from the Chart sub-menu:
SCATTER WITH ONLY MARKERS. The preview of the chart will appear with all the
points of the curve.

6. From the CHART TOOLS menu, click LAYOUT, then TRENDLINE. Click MORE
TRENDLINE OPTIONS. Check SET INTERCEPT (=0.0), DISPLAY EQUATION ON
THE CHART, and DISPLAY R-SQUARED VALUE ON CHART.

7. Click CHART TITLE, select ABOVE CHART, and type the title of the curve (e.g.,
Albumin Standard Curve, Glucose Standard Curve, etc.). Click AXIS TITLES, highlight
PRIMARY HORIZONTAL AXIS TITLE, and select TITLE BELOW AXIS to enter the
title for the x-axis (e.g., Concentration, mg/mL). For the y-axis, click AXIS TITLES
again, but this time, highlight PRIMARY VERTICAL AXIS TITLE, and select
HORIZONTAL TITLE. Enter the title for the y-axis (e.g., Absorbance,540 nm). The
legend entry may be deleted. The worksheet should now appear as follows:

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8. The "Standard Curve" graph is now completed. Print a copy.

9. To compute the concentration of the sample, use the equation of the line displayed on
the graph. Introduce the dilution factor if necessary.

For example, the equation obtained from the calibration curve is y= 11.22 x. If the
absorbance of the sample at 540 nm is 0.281, then the equation becomes 0.281 = 11.22 x.
Evaluating x produces the value 0.025 mg/mL.

HOW TO USE THE SPECTRONIC GENESYS5 SPECTROPHOTOMETER

1 Make sure that no samples are in the optical path and that the door of the sample
compartment is closed completely.

2. Plug the instrument into a 220-volt outlet.

3. Press the power switch located at the lower left side of the instrunent.

4. Let the instrument warm up for about 5 minutes. While warming up, it also diagnostic
test on its internal parts.

5. When the main menu screen appears, press 1 for absorbance or transmittance
measurement.

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6. Press GO TO WL to select the desired wavelength.

7. Enter the desired wavelength.

8. Place the blank reagent in the cell holder B and the solutions in the succeeding cell
holders.

Note: Ihe clear side of the cuvette should face the light beam and should be free from
fingerprints.

9. Use cell ʌ or cell V to move the sample into the light beam.
10. Take the reading.

11. Switch off the instrument then unplug it.

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HOW TO USE THE MULTISKAN GO MICROPLATE
SPECTROPHOTOMETER
The Multiskan GO is a spectrophotometer that can be used to measure UV/VIS
absorbance from 200 to 1000 nm on 96- or 384-well microplates. It can incubate up to
45°C and shake the microplate. Using a microplate allows for a smaller sample volume
(microliter), and up to 96 or 384 samples being read in one run. Like an ordinary
spectrophotometer, it can also be used with a cuvette.

After pressing the START/ON key, the instrument will perform self-diagnostics. (Note:
the cuvette port cover must be closed during this time). When the screen flashes the
sentence "Self-diagnostics passed," the machine is ready for use.

Procedure (Measuring with a microplate):

1. Press the IN/OUT key to bring out (open) the plate carrier. Place the microplate
containing the samples to be read on the plate carrier. Make sure that the A1 corner of
the microplate is positioned in the top left-hand corner of the carrier. Press the IN/OUT
key again to draw in (close) the plate carrier.

2. Using the keypad, enter the measurement parameters in the PLATE menu as indicated
in the experiment procedure

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3. Press the START/ON key to start the measurement.

4. Save the results of the measurement with an appropriate file name. The results may
also be exported using a USB flash drive.

5. Press the IN/OUT key to draw out the carrier and remove the microplate.

6. Switch off the instrument by pressing and holding the STOP/OFF key for a few
seconds.

Procedure (Measuring with a cuvette):

1. Using the keypad, enter the measurement parameters in the Cuvette menu.

2. Zeroing the instrument can be done with an empty cuvette port or with a cuvette with
a buffer. Press the F2 (Zero) key to zero the instrument.

3. The machine is now ready for sample reading. Insert the cuvette with the sample in the
cuvette port. Make sure that its measurement window is aligned with the measurement
direction arrow in the cuvette station.

4. Press the START/ON key to start the measurement.

5. Save the results of the measurement with an appropriate file name. The resute may
also be exported using a USB flash drive.

6. Remove the cuvette and close the port.

7. Switch off the instrument by pressing and holding the STOP/OFF key for a few
seconds.

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IMPORTANT POINTS TO CONSIDER IN UV-VIS ANALYSIS
1. Errors in UV-VIS analysis are sometimes due to the inadequate mixing of reagents.
Solutions should be well-mixed prior to absorbance reading.

2. The color formation in some assays takes place largely during the first few after
mixing. Enough time should be givern for the complete color development of the
solution before the absorbance is measured.

3. Assays should be performed in duplicate or triplicate, and the individual values not the
means, should be plotted. This procedure allows the experiment to omit erroneous values
from the calibration curve.

4. The formation of bubbles, turbidity, fingerprints, or condensation on or inside the

cuvette should be avoided because it diminishes the accuracy of the readings.

5. The "line of best fit" should be drawn through the data points. It is not necessarily the
line that passes through the origin and the other data points.

6. The Beer-Lambert's Law is most accurate between the absorbance of 0.05 to 0.70.
Above 0.70, the measured absorbance underestimates the real absorbance. Below 0.05,
the absorbance of the instrument is not accurate,

7. The calibration curve should never be extrapolated beyond the highest absorbance
value measured. If the concentration of the sample is not within the calibration curve, the
sample solution should be diluted so that its absorbance falls within the calibration curve.

APPLICATIONS OF UV-VIS SPECTROPHOTOMETRY

 Reaction kinetics
 Quantitation
 pKa determination
 DNA structural information

CENTRIFUGATION
Centrifugation is a process used to separate or concentrate materials suspended in a
liquid medium using the effect of gravity on the particles (including macromolecules) in
suspension. Two particles of different masses will settle in a tube at different rates in
response to gravity. Centrifugal force is used to increase this settling rate in an
instrument called a centrifuge.

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A centrifuge is a device used to separate the particles in a solution according to their
size, shape, density, or the viscosity of the medium and the rotor speed. A centrifuge
spins liquid samples at high speed, thus creating a strong centripetal force and causing
the denser materials to travel towards the bottom of the centrifuge tube more rapidly than
they would under the force of gravity. Centrifuges generally work under a vacuum and
are refrigerated to reduce the heating caused by frictional forces as the rotor spins. Rotors
are usually stored in refrigeration units to keep them at or near the operating temperature.

Safety Measures in Using a Centrifuge

The following precautions are based on the assumption that students have been
previously instructed on the proper use of a centrifuge or have read an instruction manual
thoroughly:

1. Make sure that the correct rotor is used and that it is installed properly on the spindle.

2. Balance the load in the rotor. Every tube must have a balance tube in the opposite slot
with the same volume of fluid.

3. Use the appropriate centrifuge tube for the experiment to avoid rupture breakage.

4. Pre-cool the centrifuge and the rotor. Rotors should be stored in a

refrigerator

5. Do not ignore the safety features of a centrifuge.

6. Never leave the centrifuge unattended until it reaches its maximum

speed or is running smoothly.

7. When in doubt, ask your laboratory instructor for help.

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