Colorimeter and

ph meterCourse: B.Sc. Biochemistry

Sem II

Sub: Biophysics and Instrumentation

Unit 3

Colorimeter

1

Introduction

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

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

• A colorimeter measures that change so users cananalyze the concentration of a particular substance inthat medium.

Beer-Lambert's law

• The device works on the basis of Beer-Lambert'slaw, which states that the absorption of lighttransmitted through a medium is directlyproportional to the concentration of the medium.

• It means that the concentration of a dissolvedsubstance, or solute, is proportional to theamount of light that it absorbs. A commonapplication of a colorimeter is therefore todetermine the concentration of a known solute ina given solution.

• A colorimeter is an instrument which comparesthe amount of light getting through a solutionwith the amount which can get through a sampleof pure solvent.

• Substances absorb light for a variety of reasons.Pigments absorb light at different wavelengths. Acloudy solution will simply scatter/block thepassage of light.

• The % transmission or the % absorbance isrecorded.

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

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

• To find the concentration of an unknown sample, severalsamples of the solution in which the concentration is known arefirst prepared and tested.

• These are then plotted on a graph with the concentration at oneaxis and the absorbance on the other to create a calibrationcurve; when the unknown sample is tested, the result iscompared to the known samples on the curve to determine theconcentration.

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.

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(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.

2

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 chemistrylaboratories, colorimeters have many practical applications.

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

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

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

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Applications

• A common application of a colorimeter istherefore to determine the concentration of aknown solute in a given solution.

• In biology, a colorimeter can be used tomonitor the growth of a bacterial or yeastculture. As the culture grows, the medium inwhich it is growing becomes increasinglycloudy and absorbs more light.

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pH meter

3

pH meter

• A pH meter is an electronic device used formeasuring the pH of a liquid (though specialprobes are sometimes used to measure the pH ofsemi-solid substances).

• A typical pH meter consists of a special measuringprobe (a glass electrode) connected to anelectronic meter that measures and displays thepH reading.

• The probe is a key part of a pH meter, it is a rodlike structure usually made up of glass. At thebottom of the probe there is a bulb, the bulb is asensitive part of a probe that contains the sensor.

The probe

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

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

• Never touch the bulb by hand and clean it with thehelp of an absorbent tissue paper with very soft hands,being careful not to rub the tissue against the glassbulb in order to avoid creating static. To measure thepH 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, whichcombines both the glass and reference electrodes into one body. Thecombination electrode consists of the following parts (see thedrawing):1. a sensing part of electrode, a bulb made from a specific glass2. internal electrode, usually silver chloride electrode or calomelelectrode3. internal solution, usually a pH=7 buffered solution of 0.1 mol/L KClfor pH electrodes4. when using the silver chloride electrode, a small amount of AgClcan 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 orcapillary 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 beingtested; (2) Glass electrode, consisting of (3) a thinlayer of silica glass containing metal salts, insidewhich there is a potassium chloride solution (4)and an internal electrode (5) made fromsilver/silver chloride. (6) Hydrogen ions formed inthe test solution interact with the outer surfaceof the glass. (7) Hydrogen ions formed in thepotassium chloride solution interact with theinside surface of the glass. (8) The metermeasures the difference in voltage between thetwo sides of the glass and converts this "potentialdifference" into a pH reading. (9) Referenceelectrode acts as a baseline or reference for themeasurement—or you can think of it as simplycompleting the circuit.

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Storage and cleaning of electrode

• Between measurements any glass and membraneelectrodes should be kept in the solution of itsown ion (Ex. pH glass electrode should be kept in0.1 mol/L HCl or 0.1 mol/L H2SO4). It is necessaryto prevent the glass membrane from drying out.

• Occasionally (about once a month), the probemay be cleaned using pH-electrode cleaningsolution; generally a 0.1 M solution ofhydrochloric acid (HCl) is used, having a pH ofone.

Calibration and use

• For very precise work the pH meter should becalibrated before each measurement. For normaluse calibration should be performed at thebeginning of each day.

• The reason for this is that the glass electrodedoes not give a reproducible e.m.f. over longerperiods of time.

• Calibration should be performed with at least twostandard buffer solutions that span the range ofpH values to be measured. For general purposesbuffers at pH 4.00 and pH 10.00 are acceptable.

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

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

• After each single measurement, the probe is rinsed withdistilled water or deionized water to remove any traces ofthe solution being measured, blotted with a scientific wipeto absorb any remaining water which could dilute thesample and thus alter the reading, and then quicklyimmersed in another solution.

Who invented the pH meter?

• First, Nobel-Prize winning German chemist FritzHaber (1868–1934) and his student ZygmuntKlemensiewicz (1886–1963) developed the glasselectrode idea in 1909.

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

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

images

1 & 2: http://www.fondriest.com/pdf/thermo_colorimeter_theory.pdf

3: http://en.wikipedia.org/wiki/PH_meter

4-7: http://www.explainthatstuff.com/how-ph-meters-work.html