INSTRUMENTATION * 0.

10 – margin of error

pH meter pH Measurement of a Sample

- Uses an electrode to measure the hydrogen ion 1. Adjust the temperature of the sample to room
concentration and pH of unknown solution temperature
- Typical pH meter is equipped with a glass electrode 2. Rinse the electrode tip with distilled water and blot dry
and a reference electrode or a single probe with tissue paper.
combination electrode connected to an electronic meter 3. Immerse the electrode in the sample solution. Allow the
that measures and displays the pH meter to stabilize then take the pH reading
- Most modern pH meters have a temperature probe for 4. Rinse the electrode tip with distilled water and blot dry
automatic temperature corrections with tissue paper.
o pH varies depending on temperature 5. Return the electrode to its filling solution.
- temp. affects the activity of - Filling solution contains a buffer that maintains the pH
hydrogen ions in glass electrode
- It can also be refilled
Glass electrode
- Consists of thin, pH-sensitive glass membrane, the inside Notes: Do’s and Don’ts
of which contains a solution with a constant 1. Calibrate the pH probe frequently to ensure accurate
concentration of hydrogen ions results.
- Contains buffer 2. Rinse the pH probe thoroughly with distilled or
Reference electrode deionized water before measuring samples.
- Does not change its voltage but sets a standard voltage 3. Store the pH probe sensing tip in soaking solution not
level to which glass electrode is compared in plain water.
- Most common: calomel electrode - The soaking solution may be pH 7 or pH 4 buffer
o Incorporates a salt bridge filled with a solution or potassium chloride (KCl) solution
saturated potassium chloride (KCl) recommended by the manufacturer
Potentiometer - Soaking solution is in the rubber
- Measures the potential across the glass membrane due 4. Do not touch the pH probe sensing tip with your bare
to concentration difference hands.
pH 5. Do not scratch or damage the pH bulb.
- Intensity or degree of acidity
- The value of pH is determined by measuring the
difference in the potential of two electrodes immersed Spectrometry
in a sample solution - Methods of analysis using spectrum
*more hydrogen ions = lower pH because pH is -log[H+] Spectrophotometry
- Measurement of an interaction between
pH electromagnetic radiation and the molecules, or atoms,
1-6 Acidic of a chemical substance
7 Neutral
8-14 Basic Colorimetric assays
- Measure the amount of light transmitted through a
pH meter: Calibration sample at a given wavelength
 pH 7.0 to 10.0, use pH 7 and pH 10 calibration buffers - Uses visible range of wavelength
- Used to determine the concentration of samples
 pH 4.0 to 7.0, use pH 4 and pH 7 calibration buffers
2 point calibration buffer – acidic and basic calibration buffers
Spectrophotometers
3 point calibration buffer – acidic, neutral and basic calibration
- Instruments that measure the amount of light absorbed
buffers
by a substance
Calibration Procedure
1. Rinse the probe tip with distilled water and blot dry
UV-Vis Spectrophotometry
with a tissue paper
- Makes the sample absorb light in the visible and near
- Use a tissue paper that does not shed fibers because it
ultraviolet region of the electromagnetic spectrum
can destroy the glass electrode
- Useful in pharmaceutical analyses
2. Place the tip of the electrode in the first buffer solution.
o Accurate and sensitive
Press “Cal”
o Has high degree of specificity
3. Take the pH reading once the reading becomes stable
- Ultraviolet – 100 to 400nm
4. Rinse the electrode tip with distilled water and blot dry
- Visible light – 400 to 700nm
with tissue paper
- Infrared – beyond 700
5. Immerse the electrode in the second buffer solution.
o Fourier-transform infrared spectroscopy (FTIR)
Press “Cal:
is a technique used to obtain an infrared
6. Take the pH reading once the pH reading become
spectrum of absorption or emission of a solid,
stable.
liquid or gas.
7. Rinse the electrode tip with distilled water and blot dry
Colors and Complementary Colors
with tissue paper.
*The pH readings obtained should correspond with the pH used
in the calibration.
 *Tungsten Lamp – light source in visible region
*Deuterium Lamp – light source in ultraviolet region

Light absorbance – based on the capacity of a solution to

absorb radiant energy.
Absorbance Concentration Transmittance
Absorbance is directly proportional to concentration but
inversely proportion with transmittance
Absorbance and transmittance is logarithmic and not linear.

Beer-Lambert’s Law
Approximate Color of Light Color of Light - determines the concentration of the absorbing species
Wavelength (nm) Absorbed Reflected in the analyte (substance of interest) solution
400-435 violet green-yellow - states that absorbance is directly related to the
435-500 blue yellow concentration if the measurements are made at a fixed
wavelength in a cell of constant path which is usually 1
500-570 green red
cm.
570-600 yellow blue - absorbance of a material sample is directly
600-630 orange green-blue proportional to its thickness
630-700 red green may be expressed by:
The complementary color of the light you see is the actual 𝐴
color. 𝐴 = Ɛ𝑐𝑙 or 𝑐 = Ɛ𝑙
Spectral Absorbance Curve
A = measured absorbance (no unit)
Ɛ = molar extinction coefficient (M-1cm-1)
c = concentration in molarity
l = path length (usually 1cm); the length of solution the light
passes through

Molar Extinction Coefficient

- a measure of how strongly a solution absorbs light in a
particular wavelength.
- If this is known, then the concentration of a solution can
be determined from the absorbance
The highest point of the curve is the most acceptable o For two solution of the same compound, one with a
wavelength to use to test the sample and know its known concentration (standard solution) and the other
concentration. with an unknown concentration, the concentration of the
unknown solution can be calculated from the measured
Spectrophotometer absorbance of the unknown solution and the
absorbance of the standard solution determined under
identical conditions, using the equation:

𝐴𝑠 𝐴𝑢 𝐴𝑢𝐶𝑠
𝐶𝑠
= 𝐶𝑢
or 𝐶𝑢 = 𝐴𝑠

Where:
Cu = concentration of the unknown solution
Cs = concentration of the standard solution
Parts Au = absorbance of the unknown solution
1. White Light Source As = absorbance of the known solution
2. Monochromator – disperse light into its compartment
wavelengths Calibration or Standard curve
3. Sample Compartment – holds the transparent tube or - preferable to construct when a large number of
cell containing the sample samples are being run
o Cuvette – holds the sample - obtained by measuring the absorbance of a series of
- Fill the cuvette ¾ full standard solutions whose concentration is in the range
- Quartz cuvette has 3mL max of the solution being measured
capacity - each standard solution must be prepared in exactly the
- Has a clear and opaque side same way as the solution being measured
 Clear side nakatapat sa *picture of a graph*
optical path because light
pass through this side Graphical Method preparation
 Opaqe side ang pwede 1. compute the concentration of the stock solution
lang mahawakan 2. make the proper dilution of the stock solution to make
4. Detector – measure the amount of light transmitted standard solutions of known concentration
5. Read-out device – display the reading 3. Read the absorbance of the standard solutions
 4. Plot the absorbance (y-axis) and the concentration of
the standard solutions (x-axis)
5. Determine the best-fit line
6. This may now be used to determine the concentration
of the sample solution
SPECTROPHOTOMETERS
 Spectronic Genesys 5
How To Use:
1. make sure that no samples are in the optical path and
that the door of the samplec 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 instrument
4. let the instrument warm up for about 5 minutes. While
warming up, it also runs a diagnostic test on its
internal parts
5. when the main menu appears, press 1 for absorbance
or transmittance measurement
6. press GO TO WL to select desired wavelength
7. place the blank reagent in the cell holder B and the
solutions in the succeeding cell holders
o the clear side of the cuvette should face the
light beam and should be free from
fingerprints
8. use cell  or  to move the sample into the light
beam
9. take the reading
10. switch off the instrument then unplug it
 Multiskan GO
- measures a UV/IS absorbance from 200 to 1000nm
on 96- or 384-well microplates
- can incubate up to 45oC and can shake the
microplate
- microplates allow up to 96 or 384 smaller sample
volumes (in microliter) to be read in one run
- can also be used with a cuvette
How To Use
1. Press the START/ON key, the instrument will perform
self-diagnostics
2. When the screen flashes “Self-diagnostics passed”,
the machine is ready to use
3. Press the IN/OUT key to bring out the plat carrier
4. Press the microplate containing the samples to be
read properly on the plate carrier
5. Pres the IN/OUY key again to close the plate carrier
6. Enter the parameters using the keypad
7. Press START/ON key to start the instrument
8. Save the result with the appropriate file name. The
file may also be exported using a USB
9. Press the IN/OUT key to draw out the carrier and
remove the microplate
10. Switch off the instrument by pressing and holding the
STOP/OFF key for a few seconds
TWO-FOLD/ SERIAL DILUTION
- A stepwise dilution method of a substance in solution
- Reduces the concentration of the substance by a factor
of two, resulting in a geometric progression (eg. ½, ¼,
1/8…) of the concentration in a logarithmic fashion
*picture*
*shake the cuvette for a homogenous mixture and change the tip
of the micropipettor for every container
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 minutes after mixing. Enough time
should be given 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 experimenter to
omit erroneous values from the calibration curve
4. The formation of bubbles, turbidity, fingerprint, or
condensation on or inside the cuvette should be
avoided because it diminishes the accuracy of the
readings
5. The “lines 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 point
6. The Beer-Lambert’s Law is most accurate between the
absorbance of 0.05 to 0.70. Above 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 diluted so that
its absorbance falls within the calibration curve
APPLICATIONS OF UV-VIS SPECTOPHOTOMETRY
 Reaction kinetics
 Quantitation
 pKa determination
 DNA structural information
The Micropipettor
- Used to transfer small amounts (<1 mL) of liquids
- Used in conjunction with disposable plastic tips
Proper Pipetting Techniques
*picture*
1. Never exceed the upper or lower limits of the pipettor
2. Set the desired volume by turning the rings clockwise to
increase volume or counterclockwise to decrease
volume
3. Place the tip on the discharge end of the pipettor
4. The plunger will stop at two different stopping points:
i. The level of depression that will result in the
desired volume of solution
ii. Used for the complete discharging of solutions
from the tip
o The second stop should not be reached when
drawing liquid into the pipettor
5. Depress the plunger until the first stopping point and
insert the tip into the solution
6. Carefully and slowly release the plunger
7. Discharge the solution into the appropriate container
by depressing the plunger to the first stop, wait one
second, and then continue pressing as far as it will go
to discharge the entire volume solution
8. Remove the tip by pressing down on the ejector button
Notes:
 Never point a pipettor up
 When withdrawing liquids, always release the
plunger slowly
 Be sure to use the proper size tip for each pipettor
  Always use a new tip for each different liquid
 Use the correct pipettor for the volume that is to
be dispensed

Solutions
- Homogenous mixture
- Composed of solute and solvent
Percentage Concentration
- “by the hundred” or “in a hundred”
- Amount of solute in a solution
- Types of percentage solutions:
o %w/w, %w/v, %v/v