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concentration.
PH = pka + log 101
Therefore PH = pka
Measurement of PH:
1. PH indicators:
An approximate idea of the PH of a solution can be obtained using indicators. These
are organic compounds of natural or synthetic origin whose colour is PH dependent.
Indicators are usually weak acids which dissociate in solution.
-
Indicators = Indicator + H+
Applying the Handerson  Hasselbath equation
-
PH = pkIn + log10 [Indicator ]
[Indicator]
The greatest colour change occurs around the pkIn and this is where the indicator is
most useful, however limitations are:
1. Colour change occurs over a wide PH range.
2. Indicators are affected by oxidizing agents, reducing agents, salt concentration,
protein etc.
A small quantity of indicator to the solution should be used, otherwise the acid base
equilibrium of the test solution may be displaced and the PH changed.
PH Meters:
The most convenient and reliable method of measuring PH is by the use of PH meter
which measures the emp. The four major parts of the PH system that are always needed
include:
1. Reference electrode
2. Indicator electrode (PH sensitive).
3. Voltmeter or amplifier
4. The sample being analyzed (fig. 11.1).
Hydrogen ion concentration is determined by the voltage that develops between the two
electrodes, the nearest equation relating electrode response to activity.
The saturated calomel electrode is usually used as the reference electrode, while the
glass electrode is used as an indicator electrode for PH measurements. Combination
electrodes (reference + indicator) are also available, being of value when the volume of
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sample is limited. Department of dairy chemistry of our college has the following instrument
for PH measurements.
1. Systronic PH meter type 331
2. Systronic PH meter type 322
3. ELICOS digital PH meter, model Li  120
4. Bechman PH meter, model H2
The operation of ELICO digital PH meter and Bechman PH meter model H2 is narrated.
ELICO digital PH meter (fig. 11.2)
This is a PH meter in which the glass and reference electrodes are combined and PH
value is displayed in digits.
1. Connects power line to mains.
2. Wash the electrode with distilled water and wipe with tissue paper.
3. Dip the electrode in standard buffer and set temp. Knob to the temp. of buffer.
4. Set PH knob to PH and adjust the PH to the above PH value of buffer.
5. Set the knob to  std  by and remove the electrode.
6. Check repeatability
7. Dip the electrodes in test solutions.
8. Repeat steps 9 & 5.
9. Wash the electrode with d/w.
Beachmen PH meter model H2:
1. Set range switch on start, connect to mains and allow to warm up for 10 mins.
2. Dip the electrodes in buffer, set temp., set proper range and adjust PH.
3. A reference point is established to correct the instrument without restandardizing.
With range switch at  nent position set the check pointer so that it coincides with
that of PH needle.
4. Rinse the electrodes with distilled water, immerse in the test solution, set proper
range (0-8 or 6 to 14), note the PH, put the switch on  nent and remove the
electrodes.
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EXERCISE 12
DEMONSTRATION OF COLORIMETER
Introduction:
Many biological experiments involve the measurement of a compound or group of
compounds present in a complex mixture colorimetry is the most widely used method
for determining the concentration of biological compound and makes the use of the
property that when light passes through a colored solution, some wavelengths are
absorbed more than others. May compounds are not colored but are made to absorb
light in the visible region by reaction with suitable reagents. The big advantage is that
complete isolation of the compound is not necessary and the constituents of complex
mixtures such as blood can be determined after little treatment.
Colorimetric techniques may be used for either qualitative or quantitative
measurements. Qualitative measurements are based on the premise that each analyte has
a unique set of energy spacing that will dictate its absorption/emission spectrum. Hence,
qualitative assays are generally based on the analysis of the absorption and/or emission
spectrum of the analyte. In contrast, quantitative assays are based on measuring the
absorbance and/or florescence of the analyte at one wavelength. Quantitative absorption
assays are based on the premise that the absorbance of the test solution will be a
function of the solution analyte concentration.
Under optimum conditions, there is a direct linear relationship between a solution s
absorbance and its analyte concentration. The equation describing this linear relationship
is known as beer s law.
Principle of operation:
Principle operation of photoelectric colorimeter type 101.
The different parts of a colorimeter are shown in figure12.1. A low voltage lamp or
energized by a constant voltage transformer forms the light source. This light passes
through a selected filter and a centrally opening shatter, controlled by a wheel, the dm of
which protrudes out. This light passes through the test-tube containing the solution and
falls on a sensitive photoelectric cell. The current generated by the photo electric cell is
amplified by a transistor amplifier which drives 50 micro ammeter calibrated in terms of
percent transmission and optical density. The transistor amplifier is in corporate mainly
to replace the delicate meter with a relatively robust 50 micro ammeter power supply.
The model of photo-electric colorimeter is shown in figure 12.1. Whereas its principle is
illustrated in figure 12.3.
Choice of filters:
Selecting the approximate filter and obtaining reproducible photometric readings are the [ Pobierz całość w formacie PDF ]