Colorimeter and

ph meterCourse: B.Sc. BiochemistrySem IISub: Biophysics and InstrumentationUnit 3

Colorimeter

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Introduction

• A colorimeter is a light-sensitive instrument that measures how much color is absorbed by an object or substance. It determines color based on the red, blue, and green components of light absorbed by the object or sample.

• When light passes through a medium, part of the light is absorbed, and as a result, there is a decrease in how much of the light reflected by the medium.

• A colorimeter measures that change so users can analyze the concentration of a particular substance in that medium.

Beer-Lambert's law

• The device works on the basis of Beer-Lambert's law, which states that the absorption of light transmitted through a medium is directly proportional to the concentration of the medium.

• It means that the concentration of a dissolved substance, or solute, is proportional to the amount of light that it absorbs. A common application of a colorimeter is therefore to determine the concentration of a known solute in a given solution.

• A colorimeter is an instrument which compares the amount of light getting through a solution with the amount which can get through a sample of pure solvent.

• Substances absorb light for a variety of reasons. Pigments absorb light at different wavelengths. A cloudy solution will simply scatter/block the passage of light.

• The % transmission or the % absorbance is recorded.

• At its most basic, a colorimeter works by passing a specific wavelength of light through a solution, and then measuring the light that comes through on the other side.

• In most cases, the more concentrated the solution is, the more light will be absorbed, which can be seen in the difference between the light at its origin and after it has passed through the solution.

• To find the concentration of an unknown sample, several samples of the solution in which the concentration is known are first prepared and tested.

• These are then plotted on a graph with the concentration at one axis and the absorbance on the other to create a calibration curve; when the unknown sample is tested, the result is compared to the known samples on the curve to determine the concentration.

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Construction• The essential parts of a

colorimeter are:– a light source, which is

usually an ordinary filament lamp

– an aperture which can be adjusted

– a detector which measures the light which has passed through the solution

– a set of filters in different colors

• filters are used to select the wavelength of light which the solution absorbs the most.

– Solutions are usually placed in glass or plastic cuvettes.

(1) Wavelength selection, (2) Printer button(3) Concentration factor

adjustment, (4) UV mode selector (Deuterium

lamp)(5) Readout(6) Sample compartment(7) Zero control (100% T), (8) Sensitivity switch.

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Advantages & Disadvantages• Advantages:

-Can be specific to one chemical species -Good for process quality control for non-chemistry personnel -Can be inexpensive per analysis

• Disadvantages:

-Similar colors from interfering substances can produce errors in results

-More precise analysis can require tighter wavelength band width (more expensive) -Matrix interferences can produce bad results in uncontrolled situations

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Uses

• Besides being valuable for basic research in chemistry laboratories, colorimeters have many practical applications.

• For instance, they are used to test for water quality, by screening for chemicals such as chlorine, fluoride, cyanide, dissolved oxygen, iron, molybdenum, zinc and hydrazine.

• They are also used to determine the concentrations of plant nutrients (such as phosphorus, nitrate and ammonia) in the soil or hemoglobin in the blood and to identify substandard and counterfeit drugs.

• In addition, they are used by the food industry and by manufacturers of paints and textiles.

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Applications

• A common application of a colorimeter is therefore to determine the concentration of a known solute in a given solution.

• In biology, a colorimeter can be used to monitor the growth of a bacterial or yeast culture. As the culture grows, the medium in which it is growing becomes increasingly cloudy and absorbs more light.

pH meter

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pH meter• A pH meter is an electronic device used for measuring

the pH of a liquid (though special probes are sometimes used to measure the pH of semi-solid substances).

• A typical pH meter consists of a special measuring probe (a glass electrode) connected to an electronic meter that measures and displays the pH reading.

• The probe is a key part of a pH meter, it is a rod like structure usually made up of glass. At the bottom of the probe there is a bulb, the bulb is a sensitive part of a probe that contains the sensor.

The probe

• The probe is a key part of a pH meter, it is a rod like structure usually made up of glass.

• At the bottom of the probe there is a bulb, the bulb is a sensitive part of a probe that contains the sensor.

• Never touch the bulb by hand and clean it with the help of an absorbent tissue paper with very soft hands, being careful not to rub the tissue against the glass bulb in order to avoid creating static. To measure the pH of a solution, the probe is dipped into the solution. The probe is fitted in an arm known as the probe arm.

Construction0f pH electrode

A typical modern pH probe is a combination electrode, which combines both the glass and reference electrodes into one body. The combination electrode consists of the following parts (see the drawing):1. a sensing part of electrode, a bulb made from a specific glass2. internal electrode, usually silver chloride electrode or calomel electrode3. internal solution, usually a pH=7 buffered solution of 0.1 mol/L KCl for pH electrodes4. when using the silver chloride electrode, a small amount of AgCl can precipitate inside the glass electrode5. reference electrode, usually the same type as 26. reference internal solution, usually 0.1 mol/L KCl7. junction with studied solution, usually made from ceramics or capillary with asbestos or quartz fiber.8. body of electrode, made from non-conductive glass or plastics. 4

How does it work?Key parts of a pH meter: (1) Solution being tested; (2) Glass electrode, consisting of (3) a thin layer of silica glass containing metal salts, inside which there is a potassium chloride solution (4) and an internal electrode (5) made from silver/silver chloride. (6) Hydrogen ions formed in the test solution interact with the outer surface of the glass. (7) Hydrogen ions formed in the potassium chloride solution interact with the inside surface of the glass. (8) The meter measures the difference in voltage between the two sides of the glass and converts this "potential difference" into a pH reading. (9) Reference electrode acts as a baseline or reference for the measurement—or you can think of it as simply completing the circuit.

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Storage and cleaning of electrode

• Between measurements any glass and membrane electrodes should be kept in the solution of its own ion (Ex. pH glass electrode should be kept in 0.1 mol/L HCl or 0.1 mol/L H2SO4). It is necessary to prevent the glass membrane from drying out.

• Occasionally (about once a month), the probe may be cleaned using pH-electrode cleaning solution; generally a 0.1 M solution of hydrochloric acid (HCl) is used, having a pH of one.

Calibration and use

• For very precise work the pH meter should be calibrated before each measurement. For normal use calibration should be performed at the beginning of each day.

• The reason for this is that the glass electrode does not give a reproducible e.m.f. over longer periods of time.

• Calibration should be performed with at least two standard buffer solutions that span the range of pH values to be measured. For general purposes buffers at pH 4.00 and pH 10.00 are acceptable.

• The pH meter has one control (calibrate) to set the meter reading equal to the value of the first standard buffer and a second control which is used to adjust the meter reading to the value of the second buffer. A third control allows the temperature to be set.

• However, for more precise measurements, a three buffer solution calibration is preferred. As pH 7 is essentially, a "zero point" calibration.

• After each single measurement, the probe is rinsed with distilled water or deionized water to remove any traces of the solution being measured, blotted with a scientific wipe to absorb any remaining water which could dilute the sample and thus alter the reading, and then quickly immersed in another solution.

Who invented the pH meter?

• First, Nobel-Prize winning German chemist Fritz Haber (1868–1934) and his student Zygmunt Klemensiewicz (1886–1963) developed the glass electrode idea in 1909.

• The modern, electronic pH meter was invented about a quarter century later, around 1934/5, when American chemist Arnold Beckman (1900–2004) figured out how to hook up a glass electrode to an amplifier and voltmeter to make a much more sensitive instrument.

Photo: How do you measure the pH of soils on Mars? Simple! You build a pH meter into a robotic space probe. The Mars Phoenix Lander space probe (left) used this built-in, mini chemical laboratory (right) to measure different aspects of the Martian soil, including acidity and metal concentrations. Photos by courtesy of NASA Jet Propulsion Laboratory (NASA-JPL).

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ReferencesReading

1. Principles and techniques of biochemistry and molecular biology by Keith Wilson, John Walker. – 7th ed.

2. http://www.explainthatstuff.com/how-ph-meters-work.html3. http://en.wikipedia.org/wiki/PH_meter4. http://www.seafriends.org.nz/dda/ph.htm5. http://www.fondriest.com/pdf/thermo_colorimeter_theory.pdf

images1 & 2: http

://www.fondriest.com/pdf/thermo_colorimeter_theory.pdf 3: http://en.wikipedia.org/wiki/PH_meter4-7:

http://www.explainthatstuff.com/how-ph-meters-work.html