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Lectures on Instrumental Analysis

Part One

For
Students of Biochemistry and analytical chemistry Diploma
Prepared by:

Dr. Kyrolus Kamal Faheem


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2010
Part One
Part one is divide into 4 main subjects: 1. Laboratory design and devices present. 2. Laboratory safety and laboratory guidelines. 3. Laboratory general techniques. 4. Chromatography.

I. Laboratory design and instruments present


A) Laboratory design and requirements:
1. The flat used as the laboratory should be of good aeration . 2. A partition in this flat should be for reception department in which people, trained for reception of patients, are present. 3. Partitions, each of which is for type of analysis e.g. microbiology analysis is in the microbiology partition and so on. 4. The main 3 persons:- A nurse (graduated from a college of nursing) who is responsible for venipuncture for collecting blood. - A well trained chemist who work as phlebotomist and analyst. - Worker who is responsible for cleaning the laboratory (chemist should know how to clean the laboratory).

B) Instruments:
1. 2. 3. 4. 5. 6. Thermostat. Oven. Balance. Magnetic stirrer. Refrigerator for preserving the samples and KITs Distillation device (distiller) to prepare distilled water.

Distillation process:
It is preferred to use double distilled deionized water in which the water is distilled twice and then pass through ion exchanger column in which any anions present in water are placed with (H+) and cations are replaced with (OH-). Tap water contains 100-250 ppm salts but the distilled water used in analysis must be almost without salts and the degree of distillation is measured by the TDS (Total Distilled Salts) and by conductivity where the distilled water doesnt conduct electricity so the less the conductivity of the distilled water, the more the distillation degree is.

Calibration:
Each device (even the balance) must be calibrated to ensure correct measurements. This is achieved by drawing a calibration curve which later is used in measuring the unknown samples.

Calibration curve: - It is a plot between the instrument signal (e.g. absorbance) and the known measured property of the sample (concentration or weight). - It is made by measuring a series of known samples each three times This lead us to a precision and accuracy.

Precision and accuracy:


They are tools to measure the degree of uncertainty in numbers obtained by measurements, observations or counting a very large quantity. Precision: - The precision of the measured numbers is the extent of agreement between repeated measurements of its value. - The precision of a number can be expressed by writing the uncertainty after a plus-or-minus sign () e.g. the uncertainty of an estimated height between 17 and 19 can be written as 181 Accuracy: It is the difference between the value of a measured number and the expected or correct value. The number is accurate only if it is close to the true value which might not be known. NOTES: The numbers can be precise, they can be accurate or they can be both. If repeated measurements give values close to one another, the numbers are precise whether or not it is accurate. Although highly precise measurements are often accurate, it is possible to measure the same incorrect number each time if the same error is made consistently, thus precision measure the extent to which measurements are reproducible.

Reproducibility of measurements:
It is the closeness of agreement among repeated measurements of a variable made under the same operating conditions over a period of time, or by different people.

II. Laboratory safety and laboratory guidelines.


Every chemist should be trained to be aware of the potential dangers of chemicals and to be careful in handling and storing chemicals. Laboratory safety should be a constant concern to everyone in the laboratory. It is your responsibility, not the instructors, to practise the laboratory safety to ensure your safety.

a) Self-protection:
1. Safety glasses or eye shields must be worn all times to guard against the laboratory accidents of others as well as your own. 2. Shoes must be worn. Wear only shoes that shed liquids. 3. Secure long hair and remove (or secure) neckties and scarves .

4. Wear nonsynthetic clothing that is not torn or frayed because in case of fires the synthetic clothes may be difficult to be removed quickly because they tend to stick to the skin. 5. Wear the laboratory coats to protect the outer clothing. 6. Never taste, smell or touch a chemical or solution unless specifically directed to do so (poisonous substances are not always labeled).

b) Personal protection

a. Goggles or safety spectacles:


Safety spectacles must be worn because eyes are vulnerable to Splashes from reagents during carrying outs any procedures where it is a risk.

b.Gloves:
Heavy duty gloves must be worn when handling both the corrosive and noncorrosive substances where the corrosive substances have obvious hazardous nature but the dangers in skin contact with noncorrosive substance are not always clear. Lightweight disposable gloves should therefore be worn during weighing and handling of chemicals to avoid the risk of absorption through the skin.

c. Protective clothing:
Laboratory coats protect the wearer from chemical splashes and infectious material. Cotton is better than nylon because cotton has a greater absorptive capacity and is generally more resistant to chemical splashes. The standard open-neck coat may be adequate for most chemical work but a high necked gown is more suitable for work with animals and potential dangerous microorganisms.

d.Face masks:
These are not always necessary but need to be worn when there is a risk of dust from chemicals or an aerosol of microorganisms.

c) Dangers to avoid:
Three rules in the laboratory must be kept to reduce the risk of poisoning resulting from the accidental transfer of a compound: a. No smoking. b. No eating and drinking. c. No mouth pipetting.

d) In case of an accident:
1. Do not panic. - The most important first action is to care the individual. - Second, take the appropriate action regarding the accident. 2. Notify the instructor even if the accident or injury is regarded as minor. 3. Wash your hands during the laboratory work and always wash your hands, arms and face before leaving the laboratory. 4. Whenever your skin (hands, arms, face, etc) comes into contact with chemicals - Wash it quickly and thoroughly with soap and water. - Use eyewash fountain to flush chemicals from the eyes and face.
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5.

6.

7. 8. 9.

- Get help immediately. - Do not rub the affected area, especially the eyes or face with your hands before washing. Chemical spills over a large part of body require immediate action: - Using the safety shower, flood the affected area for at least 5 minutes. - Remove all contaminated clothing if necessary. - Use a mild detergent and water only. - Get medical attention. Treat chemical spills in the laboratory as follow: - Alert your neighbors and the laboratory instructor. - Clean up the spill as directed by the laboratory instructor. - If the substance is volatile, flammable or toxic, warn everyone of the accident. Discharge a fire extinguisher at the base of the flames and moves it from one side to the other. For cuts, flush the affected area with water and any further treatment should be given only after consulting with the laboratory instructor. For burns, place the affected area under running water for several minutes.

e) Laboratory rules:
1. Smoking, drinking and eating including gum are not permitted at any time because chemicals may enter the mouth or lungs. (Your hands may be contaminated with an unsafe chemical). 2. The laboratory instructor must be present. 3. Unauthorized experiments are forbidden. 4. Maintain an orderly, clean laboratory desk and drawer. 5. Immediately clean up all chemical spills, paper scraps and glassware. 6. At the end of the laboratory period, completely clear the benches of equipment and clean all glassware used in the experiment. 7. Be aware of your neighbors activities because you may be a victim of their mistakes so, advise them of improper techniques or unsafe practices and if necessary tell the instructor.

f) Collecting data:
1. Record all data as they are being collected on the report sheet. 2. Record the data n ink as you perform the experiment. 3. Handle record error as follow: - If a small section of data is incorrect, cross out the incorrect data entry with a single line and clearly enter the corrected data nearby. - If a large section of data is deemed incorrect, write a short note as to why the data are in error, place a single diagonal line across the data and note where the correct data are recorded. 4. For clarity, record data entries of values less than one as 0.value e.g. 0.218 instead of .218. 5. Believe in your data (an incorrect answer resulting from honest work is infinitely better than a correct one obtained dishonorably)

g) Hazards

a. Chemical hazards:
Contact of chemicals with skin and clothing should be avoided and even if a chemical is thought to be harmless it should not be tasted or smelt. Hazard warning symbols are present on reagent bottles to warn of specific dangers and must be heeded.
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Solution of reagent placed out of the class work should also marked by the technical staff and colored adhesive labels are available for this purpose.

b.Corrosive and irritant substances:


A corrosive substance: is one that destroys living tissue e.g. strong acids or alkalis which cause very obvious injures to the skin. An irritant substance: is one that causes local inflammation but (not destruction of the tissue) results from repeated exposure e.g. some organic solvents.

c. Toxic compounds:
Compounds are graded as toxic or highly toxic depending on the dose required to kill 50% of a population of animals. Some toxic compounds take a long time before their toxicity becomes evident and this is particularly true for carcinogens (e.g. ninhydrin) and teratogens ( e.g. thyroxine)

d.Flammability hazards:
All naked flames in the laboratory should be distinguished when handling them and not only those in the immediate vicinity of the substance. Sparks from electrical equipments are less obvious than a Bunsen burner but can be just as dangerous. For this reason, organic solvents must not be stored in the refrigerator. Oxidizing substances may not be flammable themselves but may cause a fire when brought into contact with combustible material. The best precaution if such compounds need to be used, is To have only the minimum amount required on the bench and To keep the main bulk in steel cabinets well away from the work area.

e. Explosive substances:
Explosive substances are not handled in the normal biochemical laboratory but some general reagents such as picric acid used for cleaning of glasses in labs are explosive and must be handled with extreme caution. Explosions can also arise from the mixture of two compounds which in themselves are harmless. The best precaution if such compounds need to be used, is To have only the minimum amount required on the bench and To keep the main bulk in steel cabinets well away from the work area.

h) Using an Improvised Hood


The improvised hood is better than fume hood in controlling the small quantities of nauseating or toxic vapors produced because the improvised hood is connected to a water aspirator which draws the vapor from above the reaction vessel where the vapor dissolves in the water. To operate the hood completely open the faucet that is connected to the aspirator; this provides the best suction.

III. Laboratory general techniques


1) Inserting Technique Glass Tube Through a Rubber Stopper:
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NOTE: please study this technique well because many accident can occur during this technique 1. Moisten the glass tube and the hole in the rubber stopper with glycerol or water (glycerol work best). 2. Place your hand on the tube 2-3 cm from the stopper. 3. Protect your hand with a towel . 4. Simultaneously twist and push the tubing slowly and carefully through the hole. 5. Wash off any excess of glycerol on the glass and the stopper with water and dry.

2) Cleaning Glassware:
NOTE: cleanliness is extremely important in minimizing errors in the precision and accuracy. 1. Remove the residual materials with soap or detergent solution using tap water. 2. Rinse several times with tap water and then once or twice with small amounts of deionized water. 3. Discard each rinse through the delivery point of the vessel (i.e. buret tip, pipette tip, beaker spout). 4. Then wash with dilute acid (almost HCL) then with conc. acid. 5. Rinse with distilled water several times. 6. If the glass doesnt become clear (i.e. acid dont clean it well), wash with dilute base. 7. Rinse with distilled water several times. 8. Wash with volatile solution such as alcohol or acetone. 9. Then dry in the oven. If it is difficult to clean using acid and base, use the aqua regia ( mixture of 50% H2SO4 and 50% HNO3) as follow: - Pour the conc. H2SO4 slowly on the wall of the glassware, because explosion may occurs the H2SO4 mixed directly with water.

3) Handling Chemicals:
1. Read the label on the reagent bottle at least twice before removing any chemicals because the wrong chemical may lead to serious accidents or unexplainable results in your experiments. 2. Avoid using excessive amounts of reagents (never dispense more than the experiment calls for). 3. Do not return excess chemicals to the reagent bottle. 4. Never touch, taste or smell chemicals unless specifically directed to do so. 5. If inadvertent contact with a chemical occurs, wash the affected area immediately with large amounts of water.

4) Disposing of Chemicals
Discard waste chemicals as directed in the experimental procedure of each experiment or by the laboratory instructor. There are no general waste disposal containers; each is labeled for a specific waste.

5) Preparing Solutions:
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Preparation of solution starts with a reagent that is either a solid chemical or a solution more concentrated than the one recommended in the experiment. WHY? Because the concentrated solution represents the stock solution from with the required dilution will be prepared. - At either starting point, determine the minimum amount needed for the preparation which means that: For a solid: you need to know the molar mass of the compound to calculate the moles of chemical required. For a liquid: you need to know the concentration and volume (or mass) of the more concentrated solution that need to be diluted.

Precaution:
Never insert a pipette, spatula , into the reagent used for the solution preparation. Always transfer the calculated amount form the reagent bottle.

Method
Solutions are prepared from the stock solution in a volumetric flask according to the following procedure: 1. Place water (or the less concentrated solution) into the volumetric flask until it is onethird to one-half full. 2. Add the solid (or dilute the more concentrated reagent slowly, while swirling) to the volumetric flask. 3. Add enough water until the calibrated mark etched on the volumetric flask, and shake. This procedure is especially true when diluting concentrated H2SO4.

6) Measuring Mass:
Guidelines for maintain the accuracy of the balance: 1. Handle with care; balances are expensive. 2. Do not place chemicals directly on the balance pan to measure their mass but use weighing paper, a watch glass, a beaker, or some other containers. 3. Do not drop anything on the balance pan. 4. If the balance is not operating correctly, do not try to fix yourself but see your laboratory instructor. 5. Return the mass setting to zero position after completing the mass measurement. 6. Clean the balance and the balance area of any spilled chemicals. 7. If the balance is not leveled (i.e. do not return to zero position), see your laboratory instructor.

7) Using Microscale:
For safety in chemicals handling, a number of experimental procedure are designed to use small volumes of reagent solution, thus a special apparatus are needed to transfer such small volumes. There are 2 microscale apparatus: Well plates: e.g. 24 wells plate each well has the volume of about 3.5 ml. Beral pipette: it is a plastic (throwaway) pipette which has a capacity of about 2 ml.
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8) Collecting Gases
Collection of gases is classified according the solubility and density of a gas into:

f. Water-Soluble Gases, Air Displacement: g. Water-Insoluble Gases, Water Displacement:


The gas collecting flask or test tube is first filled with water, covered with a glass plate plastic wrap (no air bubbles must enter the flask) and then inverted into a pneumatic trough half-filled with water. The glass plate or plastic wrap is removed and the mouth of the flask is positioned over the gas inlet port of the pneumatic trough.

9) Transferring Solids:
Read the label on the bottle containing the solid twice to be sure it is the correct chemical

Method
1. If the bottle has a glass stopper or screw cap, place it top side down on the bench. 2. Hold the bottle with the label against your hand, tilt , and roll back and forth until the desired amount has been dispensed. 3. Estimate the amount of the solid chemical needed for the experiment and try not to dispense more reagent than that. 4. Do not return the excess to the reagent bottle, but share it with another chemist. 5. Recap the reagent bottle.

Precaution:
Do not insert a spatula or any other object into the bottle to break up the solid or to assist in dispensing the solid unless your laboratory instructor specifically advises you to do so. Do not return the excess of the chemical to the reagent bottle but share it with another chemist.

10) Transferring Liquids and Solutions:


1. Remove the glass stopper if present and hold it between fingers. 2. To transfer a liquid from one vessel to another, hold a stirring rod against the lip of the vessel containing the liquid and pour the liquid down on the stirring rod, which in turn, should touch the inner wall of the receiving vessel. 3. Recap the glass stopper to the reagent bottle.

Precaution:
Do not insert a spatula or any other object into the bottle Do not return the excess of the liquid to the reagent bottle but share it with another chemist. Never lay the glass stopper on the laboratory bench but hold it between fingers to avoid the impurities which may be picked up and contaminate the reagent.

11) Separating a liquid or solution from a solid:

a. Decanting a Liquid or Solution from a Solid:


1. Give time of the solid to settle to the bottom of the vessel. 2. Transfer the liquid (called the supernatant) with the aid of a clean stirring rod where the stirring rod is hold against the lip of the vessel containing the liquid and pour the liquid down on the stirring rod, which in turn, should touch the inner wall of the receiving vessel. Precaution: Do this slowly so as not to disturb the solid.

b.Gravity filtration:
1. 2. 3. Preparing Filter Paper for a Filter funnel (filtration): Fold the funnel as showed in the figure Place the folded filter paper snugly into the funnel. Moisten the filter paper with the solvent of the liquid/solid mixture being filtered (most likely this will be deionized water. Press the filter paper against the top of the funnel to form a seal. Support the funnel with a clump or in a funnel rack Filtrate. Precaution: The tip of the funnel should touch the wall of the receiving beaker or flask to reduce any splashing of the filtrate. The bowl of the funnel should be about half full. Always keep the funnel stem full with the filtrate because the weight of the filtrate creates a slight suction on the filter in the funnel fastening the filtration process.

4. 5. 6.

Flushing a Precipitate from the beaker: In which the solid residual stuck in the beaker is removed from the beaker with the mixtures solvent (i.e. the liquid from which the solid is separated) contained in a wash bottle, while holding the beaker over the funnel or receiving vessel, but do not wash your index finger .

c. Vacuum filtration:
In this method, Bchner funnel (a disk of filter paper over the flat, perforated bottom of the funnel) is used instead of the regular funnel because the regular funnel will rupture when vacuum is applied. Method: 1. A Bchner funnel is set unto a filter flask connected to a water aspirator which is the apparatus used form vacuum filtration.

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2. Seal the filter paper disk onto the bottom of the funnel by applying a light suction to the filter paper while adding a small amount of the solvent. 3. Once the filter is sealed, turn the water faucet attached to the aspirator completely open to create a full suction. 4. Transfer the mixture to the filter arid. 5. Wash the precipitate with an appropriate liquid.

d.Centrifugation:
A centrifuge compacts precipitate at the bottom of a centrifuge tube or test tube. The supernatant liquid is then easily decanted without any loss of precipitate. This quick separation requires 20 -40 seconds.

Precaution: 1. Never fill the centrifuge tubes to a height more than 1 cm from the top. 2. Label the centrifuge tubes to avoid confusion. 3. Always balance the centrifuge by placing an even number (i.e. 2, 4,6,) of centrifuge tubes containing equal volumes of liquid, placed opposite one another in the centrifuge. 4. This balance eliminates excessive vibration and wear. 5. If only one tube needs to be centrifuged, balance the centrifuge with a tube containing the same volume of solvent.

12) Heating Liquids and Solutions:


Liquids and solutions are heated, for examples, to promote the rate of a chemical reaction or to fasten dissolution of precipitation, in a number of different vessels. Hot liquids can be cooled by placing the glass vessel either under following tap water or in an ice bath.

a. Test Tube with a Flame


Hold the test tube with a test tube holder at a flame. Heat from top to bottom. Precaution: Test tube should be less than one-third full. Never fix the flame at the base of the test tube. Never point the test tube at anyone because the contents may be ejected violently if the test tube is not heated properly.

b.Erlenmeyer Flask with a flame


Hold the Erlenmeyer flask with flask holder not with crucible tongs or support it on wire gauze that is centered on an iron ring. Directly heat over gentle flame with gently swirling Do not place the hot flask directly on the lab bench but place it on wire gauze.

Crucible holder

Wire gauze

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c. Beaker or Flask with a Flame:


Support the beaker or the flask on wire gauze that is centered on an iron ring. Place a glass stirring rod or several boiling chips in the beaker to avoid bumping (i.e. the sudden formation of superheated liquid near the flame) - Position the flame directly under the tip of the stirring rod. - Heat slowly, occasionally agitate with the stirring rod for more uniform heating of the liquid. Precaution: Place a support iron ring at the top of the beaker or flask to avoid accidently knocking off.

d.A hot water bath


The hot water bath is used when a small quantities of liquids need to be at constant elevated temperature over a period of time. The setup is the same as that for heating a liquid in a beaker. - If the liquid is in a beaker or Erlenmeyer flask instead of a test tube, place the beaker of flask in a larger beaker, one-fourth filled with water. - Secure the beaker or flask. - Heat the water in the larger beaker to the desired temperature.

13) Evaporating a liquid:


The flammability of the liquid must be considered during liquid evaporation from a vessel. Using a fume hood or an improvised hood is recommended to remove irritating or toxic vapors.

a. Nonflammable liquid:
Using direct flame: Place the liquid in an evaporating dish centered on a wire gauze and iron ring. Slowly evaporating the liquid on a gentle flame. Using a steam bath: Place the liquid in an evaporating dish on top of a beaker Gentle boil the water in the beaker. Gentle boiling of the water in the beaker is more efficient than rapid boiling for evaporating the liquid. Avoid breathing any vapors.

b.Flammable liquid:
No open flames should be near flammable liquids. The use of a fume hood or improvised hood is suggested. Consult your laboratory instructor.

14) Heating solids


Solids are heated to dry them or test their thermal stability. Porcelain crucibles are used. Thoroughly clean the crucible before use to avoid contamination.

Complete from book pages 31-48


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VI. Chromatography
It is an analytical tool used for separation of substances from mixtures to identify and purify compounds.

1) Solution chromatography:

a. Partition Chromatography:
The separation depends upon the distribution of mixture components between the Mobile phase and liquid stationary phase According to the partition coefficient value of each constant of the mixture between the two phases. .liquid stationary phase e.g. LLC = Liquid-Liquid Chromatography (liquid mobile phase + liquid stationary phase)

b.Adsorption Chromatography:
A charge interaction occurs between the active group of the sample and the adsorbent stationary phase. - Accordingly, the components of the mixture are distributed by adsorption between a liquid mobile phase and solid adsorbent stationary phase. .solid stationary phase B stationary phase A A+B mix -: . B mobile phase A E.g. - GSC = Gas Solid Chromatography (gas mobile phase + solid stationary phase) - TLC = Thin Layer Chromatography -

2) Paper chromatography

a. Theory of paper chromatography:


1. Surface adsorption: Substance adsorbed on a filter paper by passing through the paper a solvent which would elute each substance in the mixture. 2. Ion exchange: In separating of mixtures of ions, some exchange must occur with polar constituents of cellulose and with impurities present in the paper. 3. Partition between solvents: In which partition between two immiscible phases on the filter paper occurs. The movement of a solute zone was explained as follows: - The cellulose fibers have a strong affinity to water present in solvent phase, but very little for organic liquid. - The paper acts as a stationary aqueous phase contain the solute. - Partition of the compounds occurs between the mobile organic phase and the stationary water phase. - Some of the solute leaves the paper and enter the organic phase so when the liquid reach ass section of the paper contain no solute.
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Partition occurs again, but this time solute transferred from the organic phase to paper phase. The effect of this partition is transfer of solute from point to another point take some distance along the paper in direction of solvent flow. ( )

3) Experimental technique:

a. Filter Paper:
1. Choice of filter paper: Degree and clarity of separation. Diffuseness of spots. Degree of formation of stains or streaks presumably caused by impurities in the paper. Extent of formation of tails i.e. zones other than amino acids giving color reactions with ninhydrin. - Deviation from vertical development. - Rate of movement of solvent front. 2. Modified filter paper: By modifying filter paper chemically, it may be possible for the paper to become les hydrophilic, behave like an ion exchange resin. -

b.Preparation of samples:
1. Desalting of samples: If sugars and amino acids have concentrated inorganic salts, we must desalt the sample before the chromatographic process by ion exchange treatment. 2. Ultrafiltration: For the analysis of small organic molecules in blood plasma it is essential to remove the proteins by centrifugation.

c. Application of sample Spotting:


Spots must be small and this can be using a clean capillary tubes. All spots must be in the same size.

d.Solvents development
1. Choice of solvent: The choice of a suitable solvent is governed by: - Slower moving solvents produce rounder spots and less diffused spots. - The rate of solvent movement is governed by its viscosity, surface tension and density. - It is also observed that Rf increase by raising the water content of the miscible pair of solvents. - The grade of filter paper which used for chromatography. 2. Effect of temperature and saturation: Use constant temperature because a change in temperature from one run to another will change the Rf value as the partition coefficient in the system is change. 3. Effect of chamber size - Above the critical solvent volume, the R7 values are at a minimum, even when the solvent volume is increased.
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Below the critical solvent volume, the R7 values are at a maximum.

e. Separation process chromatography:


Properties of ideal eluent: 1. The individual components of the eluent should be obtainable easily and at fairly low cost, but should be of sufficient purity for direct use. 2. It should be stable in air and when mixed with small quantities of acid. 3. It must be simple at mixing. 4. It components should be nonvolatile. 5. It should be easily removed from the sheet after the run. 6. It should be homogenous throughout the range of temperature. 7. It should not react with any of the substances to be separated. 8. The Rf values should be varies from 5 - 95 % of the sheet

f. Detection, Identification and Quantization:


1. 2. 3. 4. 5. 6. 7. 8. a. b. c. d. e. Dissolve the sample or dilute it with the suitable solvent. Spotting the sample on the base line. Choice the suitable eluent for good separation. Run the chromatograph. Detect the spot zones either by a chemical locating reagent or by U.V. light and cut each zone. Extract each zone by boiling with suitable organic solvent. Evaporate the solvent. Then, identify and quantize the separated compounds. Desirable characteristics of the locating reagents Cheap and pure. Stable alone and in solution. React rapidly with the compound in the cold. Inert towards residual solvent on the paper. It should not be a health hazard.

g.Technique for ascending paper chromatography:


For one-way chromatograms: A pencil line is drawn 2.5 cm up from the lower edge and the points of application are marked with a cross or dot. If the paper is to be cut into strips for testing with various location regents, then the number of origins should be reduced and spaced further apart. The advantages of this technique: The solution can be applied to the paper after they are mounted in the frame. Te paper is easily dried. A large number of one-way separations can be done. (i.e. more than one run can be done on the paper) This technique is very suitable for routine work and it is valuable and quick.

1. 2. 3. 4.

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4) Solvent extraction (Liquid-liquid extraction):

a. Definition:
It is a mass transfer operation in which a liquid solution (the feed) (usually water) is contacted with am immiscible or nearly miscible liquid (solvent) (usually organic solvent) that show professional affinity or selectivity towards one or more component of the feed.


It is a method to separate compounds based on their relative solubilities in two different immiscible or nearly miscible liquids, usually water and an organic solvent.

b.Products after separation:


To streams results: 1. The extract: which is the solvent rich solution containing the desired extracted solute. 2. The Raffinate: which is the residual feed solution containing little amount of the solute. EXMAPLE: If an aqueous solution (feed) of iodine and NaCl is shaken with CCl4 (organic solvent), and the liquid allowed to separate, most of the iodine will be transferred to the CCl4 layer (the extract), while the NaCl will remain in the aqueous layer (the Raffinate).

c. Equations:
Separation is governed by Nernst distribution law which state that: at equilibrium a given solute will always distributed between tow essentially immiscible liquids in the same proportions At a constant temperature and pressure: ( ) ( ) Where (KD) is the equilibrium distribution coefficient which is independent on the total concentration. If A is polarized, interacts, dissociates or form complexes with some other components of the sample or interacts with one of the solvents, the extraction process is better discussed in the terms of the terms of distribution ratio (D): ( ( ) )

d.Solvent selection:
1. 2. 3. 4. 5. High distribution coefficient. Good selectivity towards the desired solute. Has little of no miscibility with the feed solution. Should be easily recoverable for recycle. Other factors that affecting the solvent selection are boiling point, density, interfacial tension, viscosity, corrosiveness, flammability, toxicity, stability, computability with products, availability and cost.

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NOTE: Selectivity: is defined as the ability of the solvent to pick up the desired solute in the feed as compared to other solutes.

e. Selection of extraction condition:


There are 3 important conditions must be considered: 1. Temperature: Which can be used to alter selectivity where at high temperatures viscosity of the solvent become low and this can speed the solute transfer to the solvent. 2. PH: - In bio-extractions (e.g. penicillin) and agrochemicals (e.g. Orthene), PH maintained to improve distribution coefficient and minimize degradation of product. - In metal extractions, kinetic considerations govern PH. - In dissociation-based extraction of organic molecules, ph play significant role. 3. Residence time: It is an important parameter in reactive extraction processes (e.g. metals separations) and in processes involving short-life components (antibiotics & vitamins).

f. Efficiency of extraction:
Efficiency of extraction depends on the magnitude of (D) and on the relative volumes of the liquid phases. 1) (D) is large single extraction may be effect virtually quantitative transfer of solute. 2) Smaller (D) several extractions are required. 3) Extracting several times with a small volume of the organic solvent is more efficient than one extraction with a large volume. (This is significant when the volume of (D) is less than 100.

g.Method of extraction:
1. Batch extraction: - The 2 phases being shaken together in a separatory funnel until equilibrium is reached and then allowed to separate in to low layers 2. Continuous extraction: - It consists of distilling the organic solvent from a reservoir flask, condensing it and allowing it to pass through the aqueous phase before returning to the reservoir flak to be recycled. - This method is useful when D<1 and when the number of batch extractions required for quantitative transfer of solute would be large.

5) Thin Layer Chromatography:


Thin layer chromatography resembles all chromatographic techniques in: 1. All forms of chromatography have - a stationary phase which may be solid or a liquid supported on a solid - a mobile phase which may be a liquid or a gas. 2. The mobile phase flow through the stationary phase and carries the components of the mixture with it where different components travel at different rates.

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a. Application of TLC:
1. 2. 3. 4. 5. Assaying the radiochemical purity of radio pharmaceutical. Determination of plant pigments. Detection of pesticides or insecticides in food. Analyzing the dye composition of fibers in forensics. Identifying compounds present in a given substance.

b.Technique:
The stationary phase: is a thin uniform layer of silica gel or alumina coated onto a piece of glass, metals or rigid plastic and often contains a substance which fluoresce in UV light. 1. Plate preparation: a.) They are prepared by mixing the adsorbent, such as silica gel, with a small amount of inert binder like calcium sulfate (gypsum) and water. b.) This mixture is spread as thick slurry on an unreactive carrier sheet, usually glass, thick aluminum foil, or plastic. c.) The resultant plate is dried and activated by heating in an oven for thirty minutes at 110 C. d.) The thickness of the adsorbent layer is typically around 0.1 0.25 mm for analytical purposes and around 0.5 2.0 mm for preparative TLC. 2. Steps: - Suppose we start with a dye consists of simpler dyes. 1. A pencil line is drawn near the bottom of the plate and a small drop of a solution of the dye mixture is placed on it, about 1.5 centimeters from the bottom edge. 2. When the spot of the mixture dries, the plate is stood in a shallow layer of solvent in a covered beaker. 3. As the solvent slowly travels up the plate, the different components of the dye mixture travel at different rates and the mixture is separated into different colored spots. 4. The solvent is allowed to rise until almost reaches the top of the plate. That will give the maximum separation of the dye components for this particular combination of the solvent and stationary phase. 5. When the solvent front gets close to the top of the plate, the plate is removed from the beaker and the position of the solvent is marked with another line before it has a chance to evaporate. 6. Evaporate the solvent. 7. Measure the retention time (Rf) for each spot to identify each component.

Precaution 1. Any labeling on the plate to show the original position of the drop must be in pencil not in ink because dyes form ink would also moves as the chromatograph developed. 2. The solvent level must be below the pencil line. 3. The beaker must be covered to ensure that the atmosphere in the beaker is saturated with solvent vapor because this stops the solvent form evaporating as it rises up.

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3. Coloring spots: If the desired substances are colorless, there are 2 ways to color it: 1. Using Fluorescence: - The silica or alumina coated on the plate of the stationary phase often has a substance added to it which fluoresce and glow when exposed to UV light. - And thus while the UV shining on the plate, mark the positions of the spots by drawing a pencil line around them, then switch off the UV source where the spots disappears again. 2. Showing the spots up chemically: In some cases, it is possible to make the spots visible by reacting them with something which produces a colored product. For example: The spots on the chromatogram, which results from mixtures of amino acids, are colorless and can be colored by - After drying of the chromatogram, Spots are sprayed with ninhydrin which react with amino acid and form brown or purple colored compound. - After drying of the chromatogram, the chromatogram is placed in an enclosed container along with a few iodine crystals where the iodine vapor colors the spots with brown color. 4. Using TLC to identify compounds: Identifying using TLC is either with measuring Rf for each spot or by comparing the resulting chromatogram with a known chromatogram have spots of the same components of the mixture and made from previous runs. 5. Separation of compounds depends on: 1. The solubility of the compound in the solvent (mobile phase) i.e. the attraction between the molecules of the compound and those of the solvent. 2. The degree of adsorption of the compound on the stationary phase. I.e. the attraction between the molecules of the compound and those of the stationary phase. If 2 components have the same solubility in the solvent, the separation depends only on the degree of adsorption on the stationary phase and thus the separation is not efficient so try using a different solvent.

6) High Performance Liquid Chromatography, HPLC

a. Advantages of HPLC than column chromatography


1. Forcing of the solvent to flow in the column using high pressures of up to 400 atm and this makes the separation faster. 2. Use of a very much smaller particle size for the column packing material (stationary phase) which gives a much greater surface area for interactions between the stationary phase and the molecules to be separated and this make the separation better. 3. It is highly automated and extremely sensitive.

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b. Types of HPLC:
There are 2 types of HPLC depending on the relative polarity of the solvent and the stationary phase 1. Normal phase HPLC: - In which the stationary phase is polar e.g. tiny silica particles and the solvent is non-polar e.g. hexane where the non-polar compounds in the mixture, under separation process, moves more quickly than polar compounds because the polar compounds tend to stick to the polar stationary phase. - A typical column has an internal diameter of 4.6 mm or less and a length of 150 to 250 mm. 2. Reversed HPLC In which the stationary phase is non-polar (where the silica modified to be non-polar by attaching long hydrocarbon chains on its surface) and the solvent is polar e.g. mixture of water and an alcohol where the polar compounds in the mixture, under separation process, moves more quickly than non-polar compounds because the non-polar compounds tend to stick to the polar stationary phase by Van der Waals attraction forces while the polar. A typical column has an internal diameter of 4.6 mm or less and a length of 150 to 250 mm.

c. Technique:

1. Injection of the sample It is entirely automated. 2. Retention time: It is the time taken by the compound to travel through the column to the detector. This time is measured from the time at which the sample is injected to the time at which the display shows the maximum peak height for that compound. It depends on: 1. The pressure used (because that affects the flow rate of the solvent) 2. The nature of the stationary phase (not only what material it is made of, but also particle size)
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3. The exact composition of the solvent. 4. The temperature of the column. 3. The detector: Detection of the compounds occurs by determination of the absorbed UV light where the amount of light absorbed indicate the amount of the particular compound. NOTE that: solvent can absorb light so, use UV light of wavelength more than the wavelength absorbed by the solvent to avoid the false reading from the solvent.

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4. Interpreting the output from the detector: - The output is recorded as a series of peaks each one representing a compound in the mixture passing through the detector and absorb light - These peaks are used for both qualitative and quantitative analysis. 1. Qualitative analysis: It is can be done be comparing the retention time for the unknown components with a peaks of known retention time previously measured for pure samples of the various compounds under the same condition you make run on. 2. Quantitative analysis The area under each peak is proportional to the amount of the each compound and can be calculated automatically by the computer linked to the display.

d. Coupling of HPLC to a mass spectrophotometer:


In which identification of compounds is without the retention time where the detector can identify the compounds by determination of the mass during passing through it where it produce a fragmentation pattern which can be compared against a computer database of known patterns.

7) Gas Chromatography
In GC the sample is vaporized and injected onto the head of the column. Elution is brought about by the flow of an inert gaseous mobile phase. (Elution):- is continuous addition of the mobile phase to carry the solute down the column.) I) Types of gas chromatography:1. Gas liquid chromatography (GLC). 2. Gas Solid chromatography (GSC). Question:Why we must vaporize the sample (must be gas not liquid) Because gas can not transfer liquid 1. Gas Liquid chromatography (GLC) The mechanism of separation in GLC is based upon the partition of the analyte between gaseous mobile phase and a liquid stationary phase immobilized on the surface of an inert solid. GLC Instrumentation:-

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1) Carrier gas supply: Chemically inert (e.g. helium, nitrogen and hydrogen) Associated with the gas supply are pressure regulators, gauges and flow meters The choice of the gas is often according to the type of the detector. . 2) Sample injection system: We use a microsyringe to inject a liquid or gaseous sample though a self sealing, silicon rubber septum into a flash vaporizer port located at the head of the column. 3) Columns: Columns must be in oven to keep the sample in the gaseous state. There are 2 main types of columns in GC:a. Packed columns: They are made of glass, stainless steel, copper, aluminum or Teflon tubes with length of 2 to 3 m and diameter of 2 to 4 mm. These tubes are:- Densely packed with a uniform, finely divided packing material. - Densely packed with solid support, that is coated with a layer of the stationary liquid phase. b. Open Tubular Columns (capillary): There are 2 main types:1) Wall coated open tubular (WCOT):- Are simply capillary tubes coated with a thin layer of the stationary phase. - Were constructed of stainless steel, aluminum, copper, plastic or glass with an inside diameter of about 260 320 um. 2) Support coated open tubular (SCOT):Are simply capillary tubes, whose inner surface is lined with a thin film of a support material such as diatomaceous earth(solid material) NOTES: Generally the efficiency of SCOT columns is less than that of WOCT columns. The open tubular columns are more significant than that of packed columns. We must choose the stationary phase that cannot be converted into gas. 0444 stationary phase 044 . stationary 044 C 044 B 044 A A+B+C mix -: .004 044 phase The columns are formed as coils to fit in a thermostating oven. Column ovens:The optimum column temperature depends upon:1. The boiling point of the sample. 2. The degree of separation required.

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5) Detectors:There are 2 types of detectors:a. Flame Ionization Detectors (FID): First, the effluent from the column is mixed with hydrogen and air and then ignited electrically. Most organic compounds, when pyrolyzed at the temperature of a hydrogen/air flame, produce ions and electrons that can conduct electricity through the flame. A potential of a few hundred volts is applied across the burner tip and a collector electrode located above the flame. The resulting current is then directed into a high impedance operational amplifier. ionization amplifier electrode . (Effluent):- It is the liquid that passes out of the column

II) Solid support and Stationary phases used in GC a. Solid Support Materials:The solid support in a packed column serves to hold the liquid stationary phase in a place to create a large surface area as possible of at least 1 m2 /g exposed to the mobile phase.

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Properties of ideal support:1. Consists of small, uniform, spherical particles with good mechanical tTo increase the adsorption area. 2. Inert, in order not to react with sample. 3. Uniformly with the stationary liquid phase. The efficiency of a GC column increase with:1. Decreasing particle diameter the packing. 2. Higher pressure difference. b. Liquid Stationary phase in Gas Liquid Chromatographic column): Properties:1) Low volatility, In order not to vaporize easily. 2) Thermal stability, in order not to decompose at low temperature. 3) Inert, in order not to react with sample. 4) Stationary phase must show some degree of solubility of the analyte. There are 2 types of the liquid stationary phase:a. Polar Liquid Stationary Phase:e.g. polyesters - many compounds containing (-CN, -CO, and -OH) b. Non - Polar Liquid Stationary Phase:e.g. Hydrocarbons Dialkyl siloxanes The polarity of the stationary phase must match that of the sample components (Like dissolves like), this means that we will use Polar Liquid Stationary phase if we want to analyze a polar sample and non polar in case of non polar sample. When the match is good, the order of elution is determined by the boiling point of the eluents. III) Qualitative Analysis:We use chromatograph for:1) Identification of the purity of the organic compounds:The impurities (suspected compounds) or contamination of the organic compounds can be detected by the appearance of additional peaks where the areas under these peaks provide rough estimation of the extent of contamination. 2) Identification of the compounds in mixtures by measuring retention time. (Chromatogram):- is a plot of the detector signal as function of the time. (Retention time):- It is time taken after sample injection for the analyte peak to reach the detector. -:peak retention time . retention time cooking book

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IV)

Qualitative Analysis:Based on the measurement of peak height or peak area of the analyte compared with that of the standard. HOW? Prepare series of standard solutions, and then obtain the chromatogram for the standards and measure the peak height or peak area for each standard concentration. Then plot a calibration curve by plotting standard concentrations versus the peak height or peak area of each. Then obtain the chromatogram for the unknown sample and measure the peak height or area then determine the unknown sample concentration from the calibration curve.

Peak height or peak area for the unknown sample Peak height or peak area

Concentration

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