Course: BSc Biotech Sem-ISubject: Biophysics and Bioinstrumentation

Unit - 3

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

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

A colorimeter measures that change so users cananalyze the concentration of a particular substancein that medium.

The device works on the basis of Beer-Lambert's law, which states that the absorptionof light transmitted through a medium isdirectly proportional to the concentration of themedium.

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 solutein a given solution.

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

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

The % transmission or the % absorbance isrecorded.

At its most basic, a colorimeter works by passing aspecific wavelength of light through a solution, and thenmeasuring the light that comes through on the other side.

In most cases, the more concentrated the solution is, themore light will be absorbed, which can be seen in thedifference between the light at its origin and after it haspassed through the solution.

To find the concentration of an unknown sample, severalsamples of the solution in which the concentration isknown are first prepared and tested.

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

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. 7

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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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Besides being valuable for basic research in chemistrylaboratories, colorimeters have many practicalapplications.

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

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

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

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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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3

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 an electronicmeter that measures and displays the pH 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 is a key part of a pH meter, it is a rodlike structure usually made up of glass.

At the bottom of the probe there is a bulb, the bulbis a sensitive part of a probe that contains thesensor.

Never touch the bulb by hand and clean it with thehelp of an absorbent tissue paper with very softhands, being careful not to rub the tissue againstthe glass bulb in order to avoid creating static. Tomeasure the pH of a solution, the probe is dippedinto the solution. The probe is fitted in an armknown as the probe arm.

A typical modern pH probe is a combination electrode, whichcombines both the glass and reference electrodes into one body.The combination electrode consists of the following parts (seethe drawing):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 0.1mol/L KCl for pH electrodes4. when using the silver chloride electrode, a small amount ofAgCl 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 ceramicsor capillary with asbestos or quartz fiber.8. body of electrode, made from non-conductive glass orplastics.

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Key parts of a pH meter: (1) Solution beingtested; (2) Glass electrode, consisting of (3) athin layer of silica glass containing metalsalts, inside which there is a potassiumchloride solution (4) and an internalelectrode (5) made from silver/silverchloride. (6) Hydrogen ions formed in thetest solution interact with the outer surface ofthe glass. (7) Hydrogen ions formed in thepotassium chloride solution interact with theinside surface of the glass. (8) The metermeasures the difference in voltage betweenthe two sides of the glass and converts this"potential difference" into a pH reading. (9)Reference electrode acts as a baseline orreference for the measurement—or you canthink of it as simply completing the circuit.

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Between measurements any glass andmembrane electrodes should be kept in thesolution of its own ion (Ex. pH glass electrodeshould be kept in 0.1 mol/L HCl or 0.1 mol/LH2SO4). It is necessary to prevent the glassmembrane 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.

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 electrode doesnot give a reproducible e.m.f. over longer periodsof 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 themeter reading equal to the value of the first standardbuffer and a second control which is used to adjust themeter reading to the value of the second buffer. A thirdcontrol allows the temperature to be set.

However, for more precise measurements, a threebuffer solution calibration is preferred. As pH 7 isessentially, a "zero point" calibration.

After each single measurement, the probe is rinsedwith distilled water or deionized water to remove anytraces of the solution being measured, blotted with ascientific wipe to absorb any remaining water whichcould dilute the sample and thus alter the reading, andthen quickly immersed in another solution.

First, Nobel-Prize winning German chemistFritz Haber (1868–1934) and his studentZygmunt Klemensiewicz (1886–1963)developed the glass electrode 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 makea 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 MarsPhoenix Lander space probe (left) used this built-in, minichemical laboratory (right) to measure different aspects of theMartian soil, including acidity and metal concentrations. Photosby courtesy of NASA Jet Propulsion Laboratory (NASA-JPL).

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READING IMAGES

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

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

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

http://www.seafriends.org.nz/dda/ph.htm

http://www.fondriest.com/pdf/thermo_colorimeter_theory.pdf

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