instrumental methods of analysis lab manual for b. tech

INSTRUMENTAL METHODS OF ANALYSIS

LABORATORY MANUAL

DEPARMENT OF BIOTECHNOLOGY

RAJALAKSHMI ENGINEERING COLLEGE

THANDALAM, CHENNAI

Prepared by: Mrs. R. Jayasree & Mr. K.Selvaraj

Department of Biotechnology

BT 2258 INSTRUMENTAL METHODS ANALYSIS LAB MANUAL

Contents

S. No Date Name of the Experiment Sign

1 Laboratory regulations safety precautions

2 Abbreviations

3. Precision and validity in an experiment using absorption

spectroscopy and Validating Lambert-Beer's law using KMnO4

4 Precision and validity in an experiment using absorption

spectroscopy and Validating Lambert-Beer's law using

K2Cr2O7

5 Absorption spectra of Proteins in Ultra – Violet range

6 Absorption spectra of Nucleic acids in Ultra – Violet range

7 Absorption spectra of Ferrous ion by using 1, 10 -

Phenonthroline

8 Absorption spectra of Aluminium Alizarin Complex

9 Emission spectra of Aluminium Alizarin Complex

10 Estimation of Sulphate by Nephlometry

11 Separation of Plant pigments by Thin Layer Chromatography

12 Separation of leaf pigments by Column Chromatography

13 Determination of Sodium by Flame Photometry

14 Determination of Potassium by Flame Photometry

15 Glossary

16 Units, solution Preparation and Dilutions


LABORATORY REGULATIONS AND SAFETY PRECAUTIONS

General Guidelines:

1. Conduct yourself in a responsible manner at all times in the laboratory.

2. Be familiar with your lab assignment before you come to lab. Follow all written and

verbal instructions carefully. If you do not understand a direction or part of a procedure,

ask the teacher before proceeding.

3. Never work alone. No student may work in the laboratory without an instructor present.

4. When first entering a science room, do not touch any equipment, chemicals, or other

materials in the laboratory area until you are instructed to do so.

5. Do not eat food, drink beverages, or chew gum in the laboratory. Do not use laboratory

glassware as containers for food or beverages.

6. Perform only those experiments authorized by the instructor. Never do anything in the

laboratory that is not called for in the laboratory procedures or by your instructor.

Carefully follow all instructions, both written and oral. Unauthorized experiments are

prohibited.

7. Safety goggles and aprons must be worn whenever you work in lab. Gloves should be

worn whenever you use chemicals that cause skin irritations or need to handle hot

equipment. Wear older clothes that cover the maximum amount of skin.

8. Observe good housekeeping practices. Work areas should be kept clean and tidy at all

times. Bring only your laboratory instructions, worksheets, and/or reports to the work

area. Other materials (books, purses, backpacks, etc.) should be stored in the classroom

area.

9. Know the locations and operating procedures of all safety equipment including the first

aid kit, eyewash station, safety shower, spill kit, fire extinguisher, and fire blanket. Know

where the fire alarm and the exits are located.

10. Be alert and proceed with caution at all times in the laboratory. Notify the instructor

immediately of any unsafe conditions you observe.

11. Dispose of all chemical waste properly. Never mix chemicals in sink drains. Sinks are to

be used only for water and those solutions designated by the instructor. Solid chemicals,

metals, matches, filter paper, and all other insoluble materials are to be disposed of in the


proper waste containers, not in the sink. Check the label of all waste containers twice

before adding your chemical waste to the container. Cracked or broken glass should be

placed in the special container for “Broken Glass.”

12. Labels and equipment instructions must be read carefully before use. Set up and use the

prescribed apparatus as directed in the laboratory instructions provided by your teacher.

13. Keep hands away from your face, eyes, mouth, and body while using chemicals. Wash

your hands with soap and water after performing all experiments. Clean (with detergent

powder), rinse, and dry all work surfaces and equipment at the end of the experiment.

14. Experiments must be personally monitored at all times. You will be assigned a laboratory

station at which to work. Do not wander around the room, distract other students, or

interfere with the laboratory experiments of others.

15. Students are never permitted in the science storage rooms or preparation areas unless

given specific permission by their instructor.

16. Know what to do if there is a fire drill during a laboratory period; containers must be

closed, gas valves turned off, fume hoods turned off, and any electrical equipment turned

off.

17. If you spill acid or any other corrosive chemical on you skin or clothes immediately wash

area with large amounts of water (remember that small amounts of water may be worse

that no water at all). After this get the teacher’s attention. The spill kit will be used for

spills on floor or counter-top.

18. At the end of the laboratory session see that: a) main gas outlet valve is shut off b) the

water is turned off c) desk top, floor area, and sink are clean d) all equipment is cool,

clean, and arranged.

Clothing

19. Any time chemicals, heat, or glassware are used, students will wear laboratory goggles.

There will be no exceptions to this rule! Contact lenses should not be worn in the

laboratory unless you have permission from your instructor.

20. Dress properly during a laboratory activity. Long hair, dangling jewelry, and loose or

baggy clothing are a hazard in the laboratory. Long hair must be tied back and dangling


jewelry and loose or baggy clothing must be secured. Shoes must completely cover the

foot. No sandals are allowed.

Accidents and Injuries

21. Report any accident (spill, breakage, etc.) or injury (cut, burn, etc.) to the instructor

immediately, no matter how trivial it may appear.

22. If you or your lab partner is hurt, immediately yell to get the instructor's attention.

Everyone should turn off burners and prepare to help if needed.

23. If a chemical should splash in your eye(s), immediately flush with running water from the

eyewash station for at least 20 minutes. Notify the instructor immediately.

Handling Chemicals

24. All chemicals in the laboratory are to be considered dangerous. Do not touch, taste, or

smell any chemical unless specifically instructed to do so. The proper technique for

smelling chemical fumes (when instructed to do so by the teacher) is to gently fan the air

above the chemical toward your face. Breathe normally.

25. Check the label on chemical bottles twice before removing any of the contents. Take only

as much chemical as you need. Smaller amounts often work better than larger amounts.

Label all containers and massing papers holding dry chemicals.

26. Never return unused chemicals to their original containers.

27. Never use mouth suction to fill a pipette. Use a pipette bulb or pipette filler.

28. Acids must be handled with extreme care. ALWAYS ADD ACID SLOWLY TO

WATER, with slow stirring and swirling, being careful of the heat produced, particularly

with sulfuric acid.

29. Handle flammable hazardous liquids over a pan to contain spills. Never dispense

flammable liquids anywhere near an open flame or source of heat.

30. Never take chemicals or other materials from the laboratory area.

31. Take great care when transferring acids and other chemicals from one part of the

laboratory to another. Hold them securely and in the method demonstrated by the teacher

as you walk.

Handling Glassware and Equipment

32. Inserting and removing glass tubing from rubber stoppers can be dangerous. Always

lubricate glassware (tubing, thistle tubes, thermometers, etc.) before attempting to insert


it in a stopper. Always protect your hands with towels or cotton gloves when inserting

glass tubing into, or removing it from, a rubber stopper. If a piece of glassware becomes

"frozen" in a stopper, take it to your instructor for removal.

33. When removing an electrical plug from its socket, grasp the plug, not the electrical cord.

Hands must be completely dry before touching an electrical switch, plug, or outlet.

34. Examine glassware before each use. Never use chipped or cracked glassware. Never use

dirty glassware. Do not immerse hot glassware in cold water; it may shatter.

35. Report damaged electrical equipment immediately. Look for things such as frayed cords,

exposed wires, and loose connections. Do not use damaged electrical equipment.

36. If you do not understand how to use a piece of equipment, ask the instructor for help.

Heating Substances

37. SHOULD THE BUNSEN BURNER GO OUT, IMMEDIATELY TURN OFF THE

GAS AT THE GAS OUTLET VALVE. If you wish to turn off the burner, do so by

turning off the gas at the gas outlet valve first, then close the needle valve and barrel.

Never reach over an exposed flame. Light gas burners only as instructed by the teacher.

38. Never leave a lit burner unattended. Never leave anything that is being heated or is

visibly reacting unattended. Always turn the burner or hot plate off when not in use.

39. You will be instructed in the proper method of heating and boiling liquids in test tubes.

Do not point the open end of a test tube being heated at yourself or anyone else.

40. Heated metals, glass, and ceramics remain very hot for a long time. They should be set

aside to cool on wire gauze and then picked up with caution. Use tongs or heat-

protective gloves if necessary. Determine if an object is hot by bringing the back of your

hand close to it prior to grasping it.


ABBREVIATIONS

QA&QC Quality Assurance and Quality ControlLOD Limit of DetectionLLOQ Lower Limit of QuantificationULOQ Upper Limit of QuantificationFDA Food and drug administrationAUC Area under curveISO International standard for organizationHPLC High Performance liquid ChromatographySOP Standard operating procedureSTD StandardUV Ultra VioletIUPAC International union of pure and applied

chemistryGC-MS Gas chromatography – Mass SpectrumMS Mass SpectrumSRM Standard reference materialRSD Relative standard deviationSD Standard deviationIV Intravenousat. No Atomic Numberat. Wt Atomic Weightb.p Boiling PointEDTA Ethylenediamine Tetra acetic acidRF Retardation FactorESR Electron Spin ResonanceNMR Nuclear Magnetic Resonanceequiv.wt Equivalent weightGC Gas ChromatographyGLC Gas liquid ChromatographyGLP Good Laboratory PracticeGMP Good Manufacturing PracticeHPTLC High Performance thin layer chromatographyOD Optical DensityMP Melting PointIR Infra red spectroscopyTLC Thin layer chromatographyNTU Nephlo-Tubidimetric UnitPpm Parts per MillionEMR Electro Magnetic RadiationLC/MS Liquid Chromatography/Mass SpectrumFPLC Fast Protein Liquid ChromatographyPMT Photomultiplier TubeTGA Thermo gravimetric Analysis


1. VERIFICATION OF BEER-LAMBERT’S LAW

Aim: To verify Beer-Lambert’s law and to find out the molar extinction and its co-efficient or molar absorptivity.

Principle:

The Beer – Lambert’s law is a linear relationship between absorbance and concentration of an absorbing species.

Beer’s law states that “the intensity of a beam of monochromatic light decreases exponentially with increase in the concentration of absorbing species arithmetically”.

I =Intensity of incident light

C= concentration

k= Proportionality constant

-ln I = kc + b ………….. Equation 1

(On integration, b is constant of integration)

When concentration = 0, there is no absorbance. Hence I = I0

Substituting in equation 1,

-ln I0 = kX0 +b

-ln I0 = b

Substituting the value of b, in equation 1,

-ln I = kc - ln I0

ln I0-ln I = kc

ln = kc (since log A- log B = log )


ekc

e-kc

I = I0e-kc (Beer’s law)……….equation 2

Lambert’s law states that the rate of decrease of intensity (monochromatic light) with the thickness of the medium is directly proportional to the intensity of incident light.

This equation can be simplified, similar to equation 2 to get the following equation ( by replacing ‘c’ with ‘t’)

I = I0e-kt ………….equation 3

Equation 2 and 3 can be combined to get

I = I0e-kct (converting natural logarithm to base 10& K =kx0.4343)

10-Kct (rearranging term)

10Kct (inverse on the both sides)

log Kct (taking log on both sides) ………….equation 4

it can be learnt that Transmittance (T) = and Absorbance (A) =

Hence A = log

A = log …………………equation 5

Using equation 4 &5, since A = log and log = Kct we can infer that

A = Kct (instead of K, we can use ε)


A= εct (Mathematical equation for Beer – Lambert’s Law)

Where A = Absorbance or optical density or extinction co-efficient

ε = Molecular extinction coefficient

c = concentration of sample (mmol/lit)

t = path length (normality10 mm or 1 cm)

Absorbance is plotted against concentration of potassium permanganate linear relationship between absorbance and concentration of analyte is verified determining γ (gamma) value. Which indicates the extent of a given set of data and it may be obtained from the following equations.

γ =

Where N = number of pair of data x and y

x = concentration of KMnO4

y = Absorbance

Where value of γ is 1 there would be a perfect linearity lesser the value of γ from 1, lesser is the linear character of data. The variation of absorbance Vs concentration is linear and gives a straight lines

A=εct

i.e... A is proportional to c or A=εc the above equation is y=mx for m=ε

Chemicals Required:

1. Potassium permanganate

2. Distilled water

Apparatus required:

1. Spectrophotometer/ colorimeter

2. glass cuvettes

3. standard flasks, etc

Procedure:

1. A series of test tubes were labeled 1-10 (1, 2, 3…..10) respectively.


2. The stock KMnO4 solution (10 mM of KMnO4 is prepared by adding 0.158g of KMnO4 and is made up to 1000 ml) diluted into 10%, 20%, 30%, 40%............100% i.e.. KMnO4

was pipetted out into (1, 2, 3, 4….10) ml in respective tubes which was made up to 10ml by distilled water.

3. The 20%, 40% of the solution was used for the detection of λmax (i.e.) 20% and 30% o KMnO4 solution absorbance was recorded at different wavelengths ( 400nm – 800nm) using water as a blank.

4. The above selected wavelength was used for the detection of absorbance of all samples. From the correction co-efficient of KMnO4 (i.e.) γ and molar absorptivity or extinction co-efficient of KMnO4 was calculated.

Result:

The correct co-efficient (γ) of KMnO4 was equal =

The molar absorptivity or extinction co-efficient (ε) of KMnO4 was =


Wavelength selection for 20% KMnO4 solution from their maximum optical density value

S.No Wavelength Optical density1 400

2 4303 4704 4905 5206 5507 5808 6109 70010 760

Wavelength selection for 40% KMnO4 solution from their maximum optical density value

S.No Wavelength Optical density1 400

2 4303 4704 4905 5206 5507 5808 6109 70010 760

Absorbance of diluted KMnO4 sample at selected wavelength

S.No Concentration in %mM Absorbance at 520nM1 10

2 203 304 405 506 607 708 809 90


10 100

Detection of linearity (γ) absorbance Vs concentration of KMnO4


S. No

x Y xy X2 Y2

1 0.12 0.23 0.34 0.45 0.56 0.67 0.78 0.89 0.910 1.0

= = = =

Y2=

2. VERIFICATION OF BEER-LAMBERT’S LAW

Aim: To verify Beer-Lambert’s law and to find out the molar extinction and its co-efficient or molar absorptivity.

Principle:

The Beer – Lambert’s law is a linear relationship between absorbance and concentration of an absorbing species.

Beer’s law states that “the intensity of a beam of monochromatic light decreases exponentially with increase in the concentration of absorbing species arithmetically”.

I =Intensity of incident light

C= concentration

k= Proportionality constant

-ln I = kc + b ………….. Equation 1

(On integration, b is constant of integration)

When concentration = 0, there is no absorbance. Hence I = I0

Substituting in equation 1,

-ln I0 = kX0 +b

-ln I0 = b

Substituting the value of b, in equation 1,


ln I = kc - ln I0

ln I0-ln I = kc

ln = kc (since log A- log B = log )

ekc

e-kc

I = I0e-kc (Beer’s law)……….equation 2

Lambert’s law states that the rate of decrease of intensity (monochromatic light) with the thickness of the medium is directly proportional to the intensity of incident light.

This equation can be simplified, similar to equation 2 to get the following equation ( by replacing ‘c’ with ‘t’)

I = I0e-kt ………….equation 3

Equation 2 and 3 can be combined to get

I = I0e-kct (converting natural logarithm to base 10& K =kx0.4343)

10-Kct (rearranging term)

10Kct (inverse on the both sides)

log Kct (taking log on both sides) ………….equation 4

it can be learnt that Transmittance (T) = and Absorbance (A) =

Hence A = log


A = log …………………equation 5

Using equation 4 &5, since A = log and log = Kct we can infer that

A = Kct (instead of K, we can use ε)

A= εct (Mathematical equation for Beer – Lambert’s Law)

Where A = Absorbance or optical density or extinction co-efficient

ε = Molecular extinction coefficient

c = concentration of sample (mmol/lit)

t = path length (normality10 mm or 1 cm)

Absorbance is plotted against concentration of potassium permanganate linear relationship between absorbance and concentration of analyte is verified determining γ (gamma) value. Which indicates the extent of a given set of data and it may be obtained from the following equations.

γ =

Where N = number of pair of data x and y

x = concentration of KMnO4

y = Absorbance

Where value of γ is 1 there would be a perfect linearity lesser the value of γ from 1, lesser is the linear character of data. The variation of absorbance Vs concentration is linear and gives a straight lines

A=εct

i.e... A is proportional to c or A=εc the above equation is y=mx for m=ε

Chemicals Required:

1. Potassium dichromate (K2Cr2O7)

2. Distilled water

Apparatus required:



2. Glass cuvettes


Procedure:

1. A series of test tubes were labeled 1-10 (1, 2, 3…..10) respectively.

2. The stock K2Cr2O7 solution (10 mM of K2Cr2O7 is prepared by adding 0.158g of K2Cr2O7 and is made up to 1000 ml) diluted into 10%, 20%, 30%, 40%............100% i.e., K2Cr2O7

was pipetted out into (1, 2, 3, 4….10) ml in respective tubes which was made up to 10ml by distilled water.

3. The 20%, 40% of the solution was used for the detection of λmax (i.e.) 20% and 30% o K2Cr2O7 solution absorbance was recorded at different wavelengths ( 400nm – 800nm) using water as a blank.

4. The above selected wavelength was used for the detection of absorbance of all samples. From the correction co-efficient of K2Cr2O7 (i.e.) γ and molar absorptivity or extinction co-efficient of K2Cr2O7 was calculated.

Result:

The correct co-efficient (γ) of K2Cr2O7 was equal =

The molar absorptivity or extinction co-efficient (ε) of K2Cr2O7 was =


Wavelength selection for 20% K2Cr2O7 solution from their maximum optical density value

S. No Wavelength Optical density1 400

2 4303 4704 4905 5206 5507 5808 6109 70010 760

Wavelength selection for 40% K2Cr2O7 solution from their maximum optical density value

S. No Wavelength Optical density1 400

2 4303 4704 4905 5206 5507 5808 6109 70010 760

Absorbance of diluted K2Cr2O7 sample at selected wavelength

S.No Concentration in %mM Absorbance at 520nM1 10


2 203 304 405 506 607 708 809 9010 100

Detection of linearity (γ) absorbance Vs concentration of K2Cr2O7


S.no X Y XY X2 Y2

1 0.12 0.23 0.34 0.45 0.56 0.67 0.78 0.89 0.910 1.0

= = = =

Y2=

3. ABSORPTION SPECTRA OF PROTEINS IN ULTRA-VIOLET RANGE

Aim:

To obtain absorption spectra of protein in the Ultra-Violet range.

Principle:

Ultra violet spectroscopy is concerned with the study of absorption of UV radiation which ranges from 200 to 400 nm. Compounds which are colored absorb radiation from 400 to 800 nm. But compounds which are colorless absorb radiation in the UV region. In both UV as well as visible spectroscopy, only the valence electrons absorb the energy, thereby the molecule undergoes transition from ground state to excited state. This absorption is characteristic and depends on the nature of electrons present. The intensity of absorption depends on the concentration and path length as given by Beer-Lambert’s law.

The types of electrons present in any molecule may be conveniently classified as

1. ‘σ ' electrons: these are the ones present in saturated compounds. Such electrons do not absorb near UV, but absorb vacuum UV radiation (<200nm).

2. ‘π ‘electrons : These electrons are present in unsaturated compounds (eg) double or triple bonds.

3. ‘n' electrons: these are non bonded electrons which are not involved in any bonding (eg) lone pair of electrons like in S, O, N and Halogens.

Below 230 nm, the extinction of a protein solution rises steeply reaching a maximum at 190 nm, this is mainly due to the peptide bond. In practice, it is more convenient to measure the extinction at 210 nm where the specific extinction coefficient is about 200 for most proteins. All proteins here since the peptide bond content is similar.


Tyrosine and tryptophan absorb at 275 nm and 280 nm and so proteins containing these amino acids will also absorb in this region. The specific extinction coefficient varies according to how much of these amino acids are present in the particular protein.

Chemicals required:

1. Standard Bovine serum albumin

2. 9% saline solution

3. Protein in serum diluted 1: 10,000 with 9% saline solution

4. Dis.H2O

Apparatus required:

1. Spectrophotometer

2. Glass Cuvettes

3. Standard flasks, etc

Procedure:

Dissolve 10mg of Bovine Serum Albumin in 100 ml.distilled water. Add a few drops of 0.1N NaOH to preserve the solution. Prepare the following concentrations of Bovine Serum Albumin: 2mg%, 4mg%, 6mg%, 8mg%, and 10mg% as given below:

Test Tube No. 10mg% of Bovine Serum Albumin in ml.

Distilled Water in ml Concentration in mg%

1 (Blank) - 10 -2 2 8 23 4 6 44 6 4 65 8 2 86 10 0 107( Unknown) 5 5 ?

Now measure the extinction of all the solutions at 210 nm taking distilled water as blank and adjusting the spectrophotometer to 100% T with blank. Plot a graph of extinction against protein concentration (in mg %). You get a straight line graph passing through the origin. Determine the concentration of protein in the unknown solution by extrapolation.

Observations:

Test Tube No. Protein Concentration in mg% Extinction


1 -2 23 44 65 86 107 -

Calculations:

1. From Observations:

Extinction ‘A’ = 6 mg % of proteins

Extinction ’B’ = mg% of protein

1ml .of unknown solution = mg% of protein

Protein concentration in the unknown solution is= …….mg%

2. From Graph:

The concentration of protein in the unknown solution =…….mg%

Results:

1). From Calculations:

Amount of protein in the unknown sample=…….. gm%

2). From Graph:

Amount of protein in the unknown sample = ……..gm%


4. ABSORPTION SPECTRA OF NUCLEIC ACIDS IN ULTRA-VIOLET RANGE

Aim:

To obtain absorption spectra of Nucleic acid in the Ultra-Violet range.

Principle:

Ultra violet spectroscopy is concerned with the study of absorption of UV radiation which ranges from 200 to 400 nm. Compounds which are colored absorb radiation from 400 to 800 nm. But compounds which are colorless absorb radiation in the UV region. In both UV as well as visible spectroscopy, only the valence electrons absorb the energy, thereby the molecule undergoes transition from ground state to excited state. This absorption is characteristic and depends on the nature of electrons present. The intensity of absorption depends on the concentration and path length as given by Beer-Lambert’s law.

The types of electrons present in any molecule may be conveniently classified as

1. ‘σ ' electrons: these are the ones present in saturated compounds. Such electrons do not absorb near UV, but absorb vacuum UV radiation (<200nm).

2. ‘π ‘electrons : These electrons are present in unsaturated compounds (eg) double or triple bonds.

3. ‘n' electrons: these are non bonded electrons which are not involved in any bonding (eg) lone pair of electrons like in S, O, N and Halogens.


The nucleic acid absorbs strongly in UV region of spectrum due to the conjugated double bond system of the constituents purine and pyrimidine. They show characteristic maxima at 260 nm and minima at 230 nm.

Chemicals required:

1. DNA Solution

2. Saline Citrate solution

3. Dis.H2O

Apparatus required:

1. Spectrophotometer

2. glass cuvettes


Procedure:

10mg of DNA is weighed accurately and dissolved and made up to 100ml of saline citrate solution. And prepare the following concentrations: 2mg%, 4mg%, 6mg%, 8mg%, and 10mg% as given below:

Test Tube No. 10mg% of DNA Solution Distilled Water in ml Concentration in mg%1 (Blank) - 10 -2 2 8 23 4 6 44 6 4 65 8 2 86 10 0 107( Unknown) 5 5 ?

Now measure the extinction of all the solutions at 210 nm taking distilled water as blank and adjusting the spectrophotometer to 100% T with blank. Plot a graph of extinction against protein concentration (in mg %). You get a straight line graph passing through the origin. Determine the concentration of protein in the unknown solution by extrapolation.

Observations:


Test Tube No. DNA Concentration in mg% Extinction1 -2 23 44 65 86 107 -

Calculations:

1. From Observations:

Extinction ‘A’ = 6 mg % of DNA

Extinction ’B’ = mg% DNA

1ml .of unknown solution = mg% of DNA

DNA concentration in the unknown solution is= …….mg%

2. From Graph:

The concentration of DNA in the unknown solution =…….mg%

Results:

1). From Calculations:

Amount of DNA in the unknown sample=…….. gm%

2). From Graph:

Amount of DNA in the unknown sample = ……..gm%


5. ABSORPTION SPECTRUM OF FERROUS IONS BY USING 1, 10- PHENONTHROLINE.

Aim:

To determine the λmax of Fe3+ - Phenonthroline complex by using absorption spectrum.

Principle:

λmax is the wavelength at which maximum absorption of the radiation takes place. A substance may have more than one λmax . In general, increase in concentration increases absorbance of a solution but, λmax is independent of concentration i.e. λmax will not vary with change in concentration. But λmax value of a substance will vary with change in solvents. λmax value is very characteristic of a substance and hence it is used for identification of substance.

The reaction of ferrous Ammonium Sulphate and 1, 10 – Phenonthroline to form a complex which absorbs in UV and Visible region.

Chemicals required:

1. Ferrous Ammonium Sulphate – 0.02M (dissolved water (ph= 4.5) as a solvent and add 1ml Conc. Sulphuric acid)

2. 1, 10 – Phenonthroline – 0.02M (dissolved in 0.2 w/v% in alcohol).


3. Distilled water

Apparatus Required:

1. Spectrophotometer/ Colorimeter

2. Glass Cuvettes


Procedure:

1. A series of three test tubes were labeled as A (Ferrous Ammonium Sulphate), B (1, 10 – Phenonthroline), and C (Ferrous Ammonium Sulphate +1, 10 – Phenonthroline).

2. The 5ml of 0.02M Ferrous Ammonium Sulphate was pipetted out into the tube A and 5ml of 0.02M 1, 10 – Phenonthroline into tube B and pipetted out into the tube C and the contents of these tubes were mixed well with the help of cyclomixer.

3. The maximum absorbance of these solutions were recorded at different wavelengths (430, 470, 520, 550, 580, 610, 700nm) using water as a blank.

4. Three different spectral graphs were plotted (wavelength against absorption) from this λmax for Ferrous Ammonium Sulphate, 1,10- Phenonthroline and Ferrous Ammonium Sulphate- 1,10- Phenonthroline complex was calculated.

Result:

1. Maximum absorption (λmax) for Ferrous Ammonium Sulphate is………..

2. Maximum absorption (λmax) for 1, 10- Phenonthroline is………..

3. Maximum absorption (λmax) for Ferrous Ammonium Sulphate- 1, 10- Phenonthroline complex is……………

Absorption Spectrum of Ferrous Ammonium Sulphate- 1, 10- Phenonthroline complex

S.No

Wavelength Absorption of Ferrous Ammonium Sulphate

Absorption of 1, 10- Phenonthroline

Absorption of Ferrous Ammonium Sulphate- 1, 10- Phenonthroline complex

1 4302 4703 490


4 5205 5506 5807 6108 700

6. DETERMINATION OF Pka FOR PARA-NITROPHENOL

Aim:

To determine the Pka of P-Nitrophenol solution.

Principle:

The undissociated form of P-Nitrophenol in acidic medium does not absorbs radiant energy in

visible region, while quinanoid in alkaline medium absorbs strongly. The pH at which 50%

ionization or gives half of pH at which 50% ionization or gives half of the absorbance value

obtained in alkaline solution assuming 100% ionization of P-Nitrophenol.

Chemicals required:


1. Phosphate buffer ( 0.2M)

2. Carbonate-Bicarbonate buffer (0.1)

3. Citrate buffer(0.05)

4. P-Nitrophenol

Apparatus required:


2. Glass cuvettes


Preparation of buffer solution:-

Buffer 1: (Citrate Buffer)

A). Citric acid – 0.05M 210.14g/l (10.5g/l0.05M)

B). Sodium citrate- 0.05M 294.00g/l (14.70.05M)

A-145ml + B-355= 500ml (make up to 500ml) + 0.139gm (2x 10-3)

P-Nitrophenol is mixed with this buffer solution.

Buffer 2: (Phosphate Buffer)

A). KOH- 0.2M 56.11g/l (11.22g/l0.2M)

B). KH2PO4- 0.2M 136.09g/l (27.22g/l 0.2M)

A-65ml + B- 250ml + water -185ml (make up to 500ml) + 0.139gm (2x 10-3)



A).KOH- 0.2M 56.11g/l (11.2g/l0.2M)

B).KH2PO4- 0.2M 136.09g/l (27.22g/l 0.2M)

A-120ml + B- 250ml + water -130ml (make up to 500ml) + 0.139gm (2x 10-3)



A). KOH- 0.2M 56.11g/l (11.2g/l0.2M)

B). KH2PO4- 0.2M 136.09g/l (27.22g/l 0.2M)

A-200ml + B- 250ml + water -50ml (make up to 500ml)+ 0.139gm (2x 10-3)


Buffer 5: (Carbonate Buffer)

A).Sodium carbonate -0.1M 10.599g/l (10.6g/l0.1M)


B). Sodium bicarbonate -0.1M 84g/l (8.4g/l0.1M)

A-5ml + B- 465ml (make up to 500ml) + 0.139gm (2x 10-3) P-Nitrophenol is mixed with this

buffer solution.

Procedure:

1. The above prepared P-Nitrophenol obtaining buffer solutions (Citrate, Phosphate,

Carbonate).

2. Absorbances were recorded at 430nm.

3. A graph was plotted, absorbance against pH of the solutions.

4. The pH corresponding to the maximum absorbance value was divided by two to give the

Pka – value of P-Nitrophenol.

Result:

The Pka value of P-Nitrophenol is fount to be………………

Determination of Pka value for P-Nitrophenol solution

Buffer Ph Absorbance 430nm

Citrate

Phosphate I

Phosphate I

Phosphate I

Phosphate I

Calculations:


From the graph maximum pH=

Pka value of P-Nitrophenol =

7. ABSORPTION SPECTRUM OF ALUMINIUM ALIZARIN COMPLEX

Aim:

To find out the maximum absorption (λmax) of Aluminum - Alizarin complex.

Principle:

Alizarin (1, 2 dihydroxyanthroquinone) is a tricyclic aromatic diketone with two hydroxyl substituents which reacts with alum to form aluminium alizarin complex. Here covalent and coordination bonds fix the aluminium atom to 2 deprotonated alizarin molecules with the additional adjunction of water molecules.


Chemicals required:

1. Alum -0.1M

2. Alizarin – 0.001M

3. Distilled Water

Apparatus required:

1. Colorimeter

2. Test Tubes

3. Glass cuvettes

Procedure:

1. A series of three test tubes were labeled as A (Alizarin), B (Alum) and C (Alizarin+ alum).

2. The 5 ml of 0.001M alizarin was pipetted out in to the tube A and 5ml of 0.1M Alum into the B and 0.001M alizarin +0.1M alum was pipetted out into the tube C and the contents of these tubes were mixed well with the cyclomixer.

3. The maximum absorbance of these solutions was recorded at different wavelength (430nm, 470nm, 490nm, 520nm, 530nm, 580nm, 610nm, 700nm) using water as a blank.

4. Three different graphs were plotted from this λmax for alum, alizarin and alum-alizarin complex was calculated.

Result:

The maximum absorbance (λmax) of alum was…………………

The maximum absorbance (λmax) of Alizarin was…………………..

The maximum absorbance (λmax) Alizarin – Alum complex was…………….

S. No Wavelength Alum Alizarin Complex1 4302 4703 4904 520


5 5506 5807 6108 700

8. EMISSION SPECTRUM OF ALUMINIUM ALIZARIN COMPLEX

Aim:

To find out the emission spectrum for Aluminium- Alizarin complex


Principle:

Alizarin (1, 2 dihydroxyanthroquinone) is a tricyclic aromatic diketone with two hydroxyl substituents which reacts with alum to form aluminium alizarin complex. Here covalent and coordination bonds fix the aluminium atom to 2 deprotonated alizarin molecules with the additional adjunction of water molecules.

Chemicals required:

1. Alum -0.1M

2. Alizarin – 0.001M

3. Distilled Water

Apparatus required:

1. Colorimeter

2. Test Tubes

3. Glass cuvettes

Procedure:

1. A series of test tubes were labeled as 1, 2, 3, 4.

2. Alum and Alizarin were pipetted out in the series of labeled test tubes of the ratio of 1:4, 2:3, 3:2 and 4:1 respectively and the contents of the test tubes were mixed well.

3. The maximum emission of the complex solutions was recorded at 520nm (which was found in the previous experiment).

4. A graph was plotted (between ratio of alum – alizarin complex and absorbance emission spectral values) from which the maximum spectral values of the complex was detected.

Result:

The emission spectrum of alum alizarin complex was at …….ratio of alum alizarin……..


S. No Volume of AlumVolume of Alizarin

Absorbance of 520nm

1 1:42 2:33 3:24 4:1

9. ESTIMATION OF SULPHATE BY NEPHLOMETRY


Aim:

To estimate the amount of sulphate ion present in water sample by using Nephlometry.

Principle:

Nephlometry is the measurement of scattered light as a function of concentration of suspended particles (less than, approximately 100mg/liter, high concentrations).

Sulphate ions can be detected by the help of light scattering method which involves precipitation of the ions by barium chloride (BaCl2) to form a stable colloidal solution. The light scattering ability of suspension is highly dependant upon the size of particles. The amount of scattered light absorbed by photocell is directly proportional to the extent of turbidity of a solution which is observed from a digital display which is denoted as nephlometric unit.

The equation used in Nephlometry relating light scattering of a particle at specific angle of observation and concentration of solution. The intensity of transmitted light is expressed using an equation similar to that of Beer – Lambert’s law, i.e,

P =P0e-Tb

Where

P = Power of transmitted beam

P0 = Power of incident beam

T= Turbidity or turbidity Co-efficient

b = Path length

Tb = log

‘T’ was found to be proportional to the concentration (c) of suspended particles.

Hence, as T = kc, kcb = log

Wavelength:

It is expressed by the following equation.

T = S/ λt

Where


T = Turbidity

S = Constant for a given sample

λ = Wavelength

t = depends on size of particles and is ‘4’ when particle size is smaller than wavelength.

Chemicals and Equipments required:

Solution A- Hydrazine Sulphate (5gm in 400ml of distilled water)

Solution B- Hexamethylene tetramine (5gm in 400ml of distilled water)

Solution A and B were mixed and made up to 1000ml by distilled water and stored.

Working standard – 10ml of the stock was made up to 100ml by distilled water.

Test solution:

A. Sodium Sulphate (0.01M) ( 0.142gm/100ml)

B. Barium Chloride (0.01M) (0.244gm/100ml)

Apparatus:

1. Nephlo – Turbidimeter

2. Glass cuvettes

Procedure:

1. The solution A and B was mixed and made up to 1000ml by distilled water and allowed to settle for 48 hrs at room temperature to get 4000NTU.

2. Nephlometer was calibrated with formazine (Solution A + B).

3. 5ml of Sulphate solution was pipetted out into a series

4. A graph was plotted i.e., turbidence against volume of BaCl2. From the graph the equivalent point was calculated.

Result:

The amount of sulphate ion in the given sample was………..


Calculations:

Volume of Sulphate sample V1 =

Strength of Sulphate sample N1 =?

Volume of BaCl2 V2 =

Strength of BaCl2 N2 =

V1N1=V2N2

N1=

Weight of Sulphate in 1L = N1 X Eq. Wt of SO42-

Amount of SO42- ion present in 100ml of sample =………

Amount of Sulphate present in 1000ml of sample =………..mg/dl

S. No Volume of Sodium Sulphate(ml) Volume of BaCl2 Turbidence12345678910


10. SEPARATION OF PLANT PIGMENTS BY THIN LAYER CHROMATOGRAPHY

Aim:

To separate the constituents of plant pigments (leaf chlorophyll) by TLC.

Principle:

In TLC the compound under examination moves along the surface of the adsorbent. The moving substance is attracted by the polar sites on the surface of the adsorbent by electrostatic forces and this binding is reversible. The solvent also interacts with the adsorbent and the compound interacts with the solvent. This three fold competitive interaction among solute, solvent and adsorbent establishes the relative rates at which the solvent front and solute ascend the layer of adsorbent on the glass plate. A more polar solute is attracted to the adsorbent more strongly than a less polar solute. This is reflected in the faster movements of the less polar compounds.

The experiment is based on the principle of adsorption. The chlorophyll extract of plant leaves get adsorbed by the solvent molecules. The extent of adsorption of plant pigment decides partition and separation. When the solvent has reached the top of the plate, the plate is removed from the developing chamber, dried and separated components of the mixture are visualized. If the components are colored, visualization is straight forward. Usually the components are not colored so a UV lamp to visualize the plate.

Chemicals and equipments required:

1. Acetone

2. Petroleum ether

3. Mortar and Pestle

4. Developing Chamber

5. Glass plate

6. Distilled Water

Procedure:

1. Green leaves were crushed and or squashed using a mortar and pestle until a green pulp was obtained.


2. Mixture of acetone, petroleum ether (7.3), was added to dissolve the chlorophyll. The green extract was centrifuged and chlorophyll extract was obtained as a supernatant.

3. The slurry (stationary phase + water) is prepared and poured on to a glass plate which is maintained on a leveled surface. The slurry is spread uniformly on the surface of the glass plate. After setting, the plates are dried in an oven.

4. Add a sufficient mixture of solvent system (acetone, petroleum ether (7:3)) and cover the bottom of the tank to a depth of 0.5cm. Allow it to stand for 15minutes.

5. Select a TLC plate, draw a very fine line with a pencil above 1cm from one edge of the plate and apply mixtures as spot on to the plate.

6. Place the plate into the tank and allow the solvent to develop up to 15 cm.

7. Remove the plate from the tank, and allow dry it.

8. Dry the chromatogram in an oven at 60.c for 10-15 minutes. Spots will be visualized.

Result:


11. SEPARATION OF LEAF PIGMENTS BY COLUMN CHROMATOGRAPHY

Aim:

To separate the various pigments of a concentrated leaf extract using adsorption column chromatography.

Principle:

A solid stationary phase and a liquid mobile phase are used and the principle of separation is adsorption. When a mixture of components dissolved in the mobile phase is introduced in to the column, the individual components move with different rates depending upon their relative affinities. The compound with lesser affinity towards the stationary phase (adsorbent) moves faster and hence it is eluted out of the column first. The one with greater affinity towards the stationary phase (adsorbent) moves slower down the column and hence it is eluted later. Thus the compounds are separated. The type of interaction between the stationary phase (adsorbent) and the solute is reversible in nature. The rate of movement of a component (R) is given as follows:

This equation can be simplified as follows:

When a liquid mobile phase is use, the equation is written as


Where 𝛼 is the partition co-efficient

Am is the average cross section of mobile phase

As is the average cross section of stationary phase.

Requirements:

1. Chromatography (20cm X 1 cm)

2. Fresh green leaves

3. Alumina

4. Calcium carbonate

5. Sucrose

6. Sodium Sulphate

7. Petroleum ether

8. Methanol

9. Benzene

10. Mortar and pestle

Procedure:

Homogenize 5gm of green leaves in a mortar and pestle, then the extract by shaking with a mixture of petroleum ether, methanol and benzene (45: 15: 5). Remove the residue by filtration and wash the filtrate four times with water to remove the methanol. Avoid vigorous shaking or an emulsion will form. Remove the last traces of water by adding anhydrous sodium sulphate, filter to remove the solid, and concentrate the extract to a few milliliters by careful evaporation in a fume chamber.

Preparation of column:

Prepare slurries of the column materials ( alumina, calcium carbonate and sucrose) in petroleum ether and pack the column with alumina (5cm) ,calcium carbonate (7cm) and sucrose(7cm), inserting a filter paper disc between each adsorbent . Gentle suction may be applied to the bottom of the column to assist packing. Wash the column with several volumes of the eluting solvent, a mixture of benzene and petroleum ether (1:4).


Separation and elution of the pigments:

When the top of the column is almost dry, add the extract and elute the solvent. If the flow rate is too slow, apply gentle pressure to the top of the column.

Observation:

Column details:

Column adsorbent:

Column height:

Eluting agent:

Flow rate:

Numbers of bands………………..

Result:

The number of bands obtained in this experiment was…………….

Note:

The color chart for the pigments is as follows:

Chlorophyll ‘a’ and chlorophyll ‘b’ – green

Xanthophylls- yellow

Carotenes – orange

By continuously adding the eluting solvent, collect the factions and plot the absorption spectrum of each colored peak.


12. DETERMINATION OF SODIUM BY FLAME PHOTOMETRY

Aim:

To determine the amount of sodium present in the given sample solution.

Principle:

Flame emission spectroscopy is a type of atomic emission spectroscopy. It is mostly applicable for analysis of alkali and alkali earth metals. In this spectroscopy, the sample solution of sodium salt is nebulized in to flame, which may produce solid residue upon solvent evaporation. This solid residue undergoes atomization and gives neutral atoms which may acquire thermal energy from flame and undergoes electronic excitation. Due to unstable nature of excited state, excited atoms come back to ground state by emission of absorbed energy as visible radiation. By measuring the wavelength and intensity of emitted radiation, we can do qualitative and quantitative analysis respectively.

Chemicals required:

1. Sodium chloride

2. Distilled water

Apparatus required:

1. Flame photometer


2. Volumetric flasks

3. Pipette

Procedure:

Preparation of standard solutions for calibration curve:

Dissolve exactly 2.542gm of sodium chloride in water and make up to 1 liter. This contains 0.1mg per ml (1000) ppm. Dilute this to 10, 5, 2.5, and 1 ppm sodium ion solutions.

Estimation of sodium by flame photometer:

First, switch on the digital flame photometer followed by the air compressor with the required value (10 bar)

Open the gas from the gas cylinder (after the instrument is warmed up for 10 minutes). Initially allow the ion-free water (distilled water) to aspirate in to the flame and set the digital value as 100. Now the instrument is said to be calibrated. After this calibration of the instrument, no adjustment should be made .Introduce the solutions containing different concentrations of sodium chloride (2, 4, 6, 8, 10µg) to the flame and find out the intensity of emitted light of each solution.

Plot a calibration graph between concentration and intensity of NaCl solution which passes through the origin. Finally, introduce the sample of unknown solution containing sodium into the flame and find out the intensity of emitted radiation. From the intensity, the concentration of unknown solution can be determined.

Result:

The amount of sodium in the given sample………..ppm

Determination of sodium

S. No Concentration of NaCl Solution(ppm) Flame intensity1 102 83 64 45 26 unknown


The concentration of unknown sample =

13. DETERMINATION OF POTASSIUM BY FLAME PHOTOMETRY

Aim:

To determine the amount of potassium present in the given sample solution.

Principle:

Flame emission spectroscopy is a type of atomic emission spectroscopy. It is mostly applicable for analysis of alkali and alkali earth metals. In this spectroscopy, the sample solution of sodium salt is nebulized in to flame, which may produce solid residue upon solvent evaporation. This solid residue undergoes atomization and gives neutral atoms which may acquire thermal energy from flame and undergoes electronic excitation. Due to unstable nature of excited state, excited atoms come back to ground state by emission of absorbed energy as visible radiation. By measuring the wavelength and intensity of emitted radiation, we can do qualitative and quantitative analysis respectively.

Chemicals required:

1. Potassium chloride


2. Distilled water

Apparatus required:

1. Flame photometer

2. Volumetric flasks

3. Pipette

Procedure:

Preparation of standard solutions for calibration curve:

Dissolve exactly 1.090gm of potassium chloride in water and make up to 1 liter. This contains1mg per ml (1000 ppm).

Estimation of potassium by flame photometer:

First, switch on the digital flame photometer followed by the air compressor with the required value (10 bar)

Open the gas from the gas cylinder (after the instrument is warmed up for 10 minutes). Initially allow the ion-free water (distilled water) to aspirate in to the flame and set the digital value as 100. Now the instrument is said to be calibrated. After this calibration of the instrument, no adjustment should be made .Introduce the solutions containing different concentrations of potassium chloride (2, 4, 6, 8, 10µg) to the flame and find out the intensity of emitted light of each solution.

Plot a calibration graph between concentration and intensity of KCl solution which passes through the origin. Finally, introduce the sample of unknown solution containing sodium into the flame and find out the intensity of emitted radiation. From the intensity, the concentration of unknown solution can be determined.

Result:

The amount of potassium in the given sample………..ppm

Determination of Potassium

S. No Concentration of KCl Solution(ppm) Flame intensity1 102 83 64 4


5 26 Unknown

The concentration of unknown sample =

Glossary:

Analysis: Analysis deals with methods for determining the chemical composition of samples of matter.

Accuracy: Accuracy describes the correctness of an experimental result. Accuracy is expressed in terms of either absolute error or relative error.

Absolute error= Observed value – True value

Analyte: A specific chemical moiety being measured, this can be intact drug, bimolecular or its derivative, metabolite, and/ or degradation product in a biologic matrix.

Absorbance: when light passes through a solution, light of certain wavelength is absorbed by the colored chemical substance and it is referred to as the absorbance, and the remaining light is


transmitted which is referred to as transmittance; absorbance is also known as optical density (OD) or extinction.

Biological matrix: a discrete material of biological origin that can be sampled and processed in a reproducible manner. Examples are blood, serum, plasma, urine feces, saliva, sputum and various discrete tissues.

Blank: A sample of a biological matrix to which no analytes have been added that is used to assess the specificity of the bioanalytical method.

Beer’s Law: the intensity of a beam of monochromatic light decreases exponentially with increase in the concentration of absorbing species arithmetically.

Band Pass: Band pass is the difference in wavelength between the points where the transmittance is one – half the maximum.

Bias: bias provides a measure of the systemic, or determinate, error of an analytical method. Bias is defined by the equation

Bias = µ - xt

Where µ is the populations mean for the concentration of an analyte in a sample that has a true concentration of xt.

Calibration standard: a biological matrix to which a known amount of analyte has been added or spiked. Calibration standards are used to construct calibration curves from which the concentration of analytes in QCs and in unknown study samples are determined.

Chromatography: is the separation of a mixture in to individual components using stationary phase and a mobile phase.

EMR: electro magnetic radiation is made up of discrete particles called photons.

Error: the error of a measurement is an inverse measure of its accuracy. The similar, the error, greater is the accuracy of the analysis.

Errors can be expressed as either absolute error or relative error

Absolute error= observed error –true value.

Relative error = x 100%

Frequency: frequency is the number of complete wavelength units passing through a given point in unit time. Frequency is measured in HZ (Hertz) or cps (cycles per second).


Lamberts Law: the rate of decrease of intensity (monochromatic light) with the thickness of the medium is directly proportional to the intensity of incident light.

Limit of detection (LOD): the lowest concentration of an analyte that the bioanalytical procedure can reliably differentiate from background noise.

Lower limit of quantification (LLOQ): the lowest amount of an analyte in a sample that can be quantitatively determined with suitable precision and accuracy.

Monochromatic light: light have single wavelength.

Nephlometry: is the measurement of scattered light as a function of concentration of suspended particles (less than, approximately 100mg/liter)

Polychromatic light: the light radiations wavelength has 0 to 1000nanometers

Precision: Precision describes the reproducibility of results. Reproducibility of results is the agreement between numerical values for two or more replicate measurements. In other words, it is the agreement between measurements that have been made in exactly the same way. The three terms which are used to describe the precision of a set of replicate data.

1. Standard deviation: it is the root mean square deviation from mean

2. Variance: square of standard deviation

3. Co-efficient of variation x 100%

Sample: a generic term encompassing controls, blanks, unknowns, and processed samples.

Spectroscopy: is the measurement and interpretation of electromagnetic radiation (EMR) absorbed or emitted when the molecules or atoms or ions of a sample move from one energy state to another energy state. This change may be from ground state to excited state or excited state to ground state. At ground state, the energy of a molecule is the sum total of rotational, vibrational and electronic energies.

Sensitivity: According to IUPAC, a method is said to be sensitive if small changes in concentration causes large changes in the response function. Sensitivity can be expressed as the slope of the linear regression calibration curve, and it is measured at the same time as the linearity tests. The sensitivity attainable with an analytical method depends on the nature of the analyte and the detection technique employed. The sensitivity required for a specific response depends on the concentrations to be measured in the biological specimens generated in the specific study.


Internal standard: an internal standard is a substance that is added in a constant amount to all samples, blanks, and calibration standards in an analysis.

Turbidimetry: is the measurement of transmitted light as a function of concentration of suspended particles (more than 100mg / liter, high concentration)

Wave length: wavelength is the distance between two successive maxima or minima or distance between two successive troughs or peaks. Wavelength can be measured in meters, centimeters (cm or10-2), millimeters (mm or 10-3), micrometers (µ or 10-6), nanometers (nm or 10-9).

Wave number: it is the number of waves per cm.

Wave number is expressed in cm-1 or Kayser.

Unknown: a biological sample that is the subject of the analysis.

Upper limit of quantification (ULOQ): the highest amount of an analyte in a sample that can be quantitatively determined with precision and accuracy.

Qualitative analysis: a qualitative method yields information about the identity of atomic or molecular species or the functional groups in the sample.

Quantitative analysis: a quantitative method, in contrast, provides numerical information as to the relative amount of one or more of these components.

UNITS, SOLUTION PREPARATION AND DILUTIONS

For Wavelength:

1 meter = 100 centimeter (cm) or 1000 millimeter (mm)

1 millimeter = 1000 microns (µm)

1 µm = 1000 nanometer (nm)

1nm = 1000 picometer (pm)

1 mm = 10-3m


1 µm = 10-6m

1nm = 10-9m

1Å =10-10m

1pm = 10-12m

The visible Spectrum:

Wavelength region, nm

Color Complementary Color

400 -435 Violet Yellow - green435-480 Blue Yellow480-500 Blue-green Red500-560 Green Purple560-580 Yellow-Green Violet580-595 Yellow Blue595-650 Orange Green-blue650-750 Red Blue-green

For Frequency:

1 Kilo Hertz = 103 Hz

1 Mega Hertz = 106 Hz

1 Giga Hertz = 10 9 Hz

1 Tera Hertz = 10 12 Hz

For weight:

1 Kilogram = 1000 gram (gm)

1 gram = 1000 milligram (mg)

1 milligram = 1000 microgram (µg)

1 microgram = 1000 nanogram (ng)

1mg =10-3g

1 µg = 10-6g

1ng = 10-9g


Percentage solution and conversion:

1% w/v = 1gram in 100ml solvent

1% w/w = 1 gram in 100 grams of solvent

Normally %w/v solutions are used

Conversions:

1. To convert % w/v to mg/ml : multiply 10

%w/v x 10 = mg/ml

Problem: convert 2% w/v to mg/ml

Formula: 2% w/v x 10 = 20mg/ml

Answer is 20mg/ml

(2% w/v means 2grams/ 100 ml

i.e . 2000mg/100ml

ie. 20mg/ml)

2. To convert mg/ml to %w/v: divide by 10

Problem: convert 30mg/ml to %w/v

Formula:

Answer is 3% w/v

(Checking: 1 ml contains 30mg

100ml contains 30x 100mg = 3000mg= 3gm

i.e 3gm/100 ml = 3% w/v)

3. To convert %w/v to µg/ml: multiply by 10,000

%w/v X 10000= µg/ml


Problem: convert 2%w/v to µg/ml

Formula: 2%w/v x 10,000= 20,000 µg/ml

(2%w/v means 2grams /100ml

i.e., 2000mg in 100ml= 20mg/ml = 20000 µg/ml)

4. What is the percent concentration of a solution that you made by taking 5.85 g of NaCl

and diluting to 100 ml with H20?

5.85 g/100 ml = 5.85% W/V solution of NaCl

5. What is the percent concentration of a solution that you made by taking 40 g of CaCl2 and diluting to 500 ml with H20?

You set up a proportion problem.

40 g /500 ml = Xg /100 ml

X = 8g

8g/100 ml = 8% (W/V) solution

OR another way to look at this is, 40 grams solute is what percent of the 500 mL solution? The 100 is used to convert to percent.

6. How would you make 250 ml of a 8.5% NaCl solution?

This works backwards from the others --

8.5%= 8.5 g /100 ml

Again, set up a proportion

8.5 g /100 ml= X / 250 ml

21.3 g = X

OR an alternative method is to say what 8.5% of 250 ml is?

250 x 0.085 = 21.3

Therefore you would need to weigh out 21.3 g NaCl and dilute to 250 ml with H20.


7. How much (volume) 0.85% NaCl may be made from 2.55 g NaCl?

An 0.85% NaCl solution = 0.85 g/100 ml

Setting up a proportion again,

0.85 g /100 ml = 2.55 g/X

X = 300 ml

Therefore, 300 ml of 0.85% NaCl may be made from 2.55 g NaCl.

MOLAR SOLUTIONS (M)

The definition of molar solution is a solution that contains 1 mole of solute in each liter of solution. A mole is the number of gram molecular weights (gmw). Therefore, we can also say a 1M = 1 g MW solute/liter solution.

1M NaCl solution would be

Na = MW of 23

Cl = MW of 35.5

NaCl = MW of 58.5

1M = 58.5 g of NaCl in 1 liter of solution.

It may be made by weighing out 58.5 g of NaCl and qs to 1 liter with water. The qs stand for quantity sufficient and are a term used to designate that the total volume must be 1 liter (or whatever is stated).

58.5 g NaCl qs 1 liter with H20

Examples of other solutions would be

1 M H2S04 = 98 g/L

1 M H3P04 = 98 g/L

PROBLEMS

1. How would you make a liter of 4M CaCl2?

First find molecular weights.


Ca = 40

Cl2 = 35.5 x 2 = 71

CaCl2 = 111 (MW)

Then,

1M = 111 g/L

4M = 4 (111 g/L)

= 444 g/L

Weigh out 444g CaCl2 qs 1 liter with H20

2. How would you make 300 ml of a 0.5M NaOH solution?

First find molecular weights.

Na = 23

0 = 16

H = 1

NaOH = 40 (MW)

Then,

1M = 40 g/L

0.5M = 0.5 (40 g/L)

= 20 g/L

But you only want 300 ml so . . .

20 /1000 ml = x /300 ml

6 g = x

Weigh out 6 g NaOH pellets qs. 300 ml with H20


NORMAL SOLUTIONS:

The definition of a normal solution is a solution that contains 1 gram equivalent weight (gEW) per liter solution. An equivalent weight is equal to the molecular weight divided by the valence (replaceable H ions).

1N NaCl = 58.5 g/L

1N HCl = 36.5 g/L

1N H2S04 = 49 g/L

Problems involving normality are worked the same as those involving molarity but the valence must be considered:

1N HCL the MW= 36.5 the EW = 36.5 and 1N would be 36.5 g/L

1N H2SO4 the MW = 98 the EW = 49 and 1N would be 49 g/L

1N H3PO4 the MW = 98 the EW = 32.7 and 1N would be 32.7 g/L

PROBLEMS:

1. You weigh out 80 g of NaOH pellets and dilute to 1 liter. What is the normality?

MW of NaOH = 40

EW = 40

1N = 40 g/L

80 g/L /40 g/L = 2N

What is the molarity?

MW = 40

1M = 40 g/L

80 g/L /40 g/L = 2M

2. You weighed out 222g of CaCl2 and diluted to 1 liter. What is the normality?

EW = 111 /2 = 5.55

1N = 55.5 g/L


222 g/L /55.5 g/L = 4N

What is the molarity?

1M = 111 g/L

222 g/L /111 g/L = 2M


instrumental methods of analysis lab manual for b. tech

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