Dr. Abdul Muttaleb Jaber Professor of Analytical Chemistry Email: ajaber@philadelphia.edu.jo

0510113 Pharmaceutical Chemical Analysis

Textbook

Analytical Chemsitry

Gary D. Christian

6th Edition, 2004

Chapter 1

Analytical objectives or what analytical chemists do?

What is analytical chemistry?

• Analytical chemistry is the science that deals with the chemical characterization (qualitative and quantitative) of matter.

• Analytical chemistry is applied in many aspects of chemistry: agricultural, clinical, environmental, forensic, manufacturing, metallurgical, and pharmaceutical chemistry.

• The quality of manufactured products depends on proper chemical proportions of active components, and measurement of these components is the basis of the quality control/assurance of products.

• Qualitative analysis deals with the identification of elements, ions, or compounds present in a sample.

• Quantitative analysis deals with the determination of how much of one or mo re constituents is present.

Classifying Analytical Methods

1. Wet methods of chemical analysis (Traditional or classical methods):

• Gravimetric Methods: The analyte is precipitated by a certain reagent. The amount of the analyte is determined from the mass of the precipitate

• Volumetric Methods: the concentration of the analyte is determined as a result of adding to it a measured volume of solution containing a reagent of known concentration to react completely with it.

Analytical Methods

2. Instrumental Methods of analysis • Electroanalytical Methods: it involve the measurement of

voltage, current, resistance, or quantity of electrical charge. • Spectroscopic Methods: it involves the interaction between

electromagnetic radiation and matter (analyte atoms or molecules).

• Chromatographic methods: it is based on separating the components of a mixture as a result of their degree of distribution between two phases (mobile and stationary phase)

• Miscellaneous Methods: include rate of radioactive decay, heat of reaction, rate of reaction, thermal conductivity, optical activity, refractive index, and others

THE ANAL YTICAL PROCESS

The analytical process may be take place according to the following sequence of events:

(1) defining the problem

(2) obtaining a representative sample

(3) preparing the sample for analysis

(4) performing necessary chemical separations

(5) performing the measurement

(6) calculating the results and presenting the data.

Fig. 1.1. Steps in an analysis

An analysis involves several

steps and operations which

depend on:

•the particular problem

• your expertise

• the apparatus or

equipment available.

The analyst should be

involved in every step.

©Gary Christian,

Analytical Chemistry,

6th Ed. (Wiley)

Defining the problem

• Before the analyst can design an analysis procedure, he / she must know

• what information is needed? • what type of sample is to be analyzed • how the sample is to be obtained • how much of the sample is needed • how sensitive the method must be • how accurate and precise it must be? • what separations may be required to eliminate

interferences.

Acquiring the Sample

• A laboratory analysis sample must be representative of the whole so that the final result of the chemical analysis represents the entire system

• The material to be sampled may be solid, liquid, or gas. It may be homogeneous or heterogeneous in composition.

– In the homogeneous case, a simple "grab sample" taken at random will suffice for the analysis.

– In the heterogeneous case, we may be interested in the variation throughout the sample, in which case several individual samples will be required.

Processing the Sample

• This step may or may not be necessary, depending upon the type of sample

• The first step in processing the sample is the preparation of a laboratory sample

• Preparing a Laboratory Sample

– A solid laboratory sample is ground to decrease particle size, mixed to ensure homogeneity, and stored for various lengths of time before analysis begins

– A Liquid samples should be prepared as not to allow evaporation because that can change the concentration of the sample

Replicate Samples

– Most chemical analyses are performed on replicate samples whose masses or volumes have been determined by careful measurements with an analytical balance or with a precise volumetric device

– Replicate samples are portions of a material of approximately the same size that are carried through an analytical procedure at the same time and in the same way

Replication improves the quality of the results and provides a measure of their reliability through statistical analysis

Preparing the sample for analysis

• The vast majority of all analysis procedures call for the sample to be in solution

• Sometimes this will mean complete dissolution of the entire sample

• Laboratory reagents and solvents must be employed for this purpose.

• Examples of these reagents are:

Substance Description Uses

Water Clear, colorless liquid with Dissolving polar and ioni

H20 low vapor pressure compounds ( ionic salts)

highly polar.

Hydrochloric Commercially available as Dissolving

acid, HCl 38% water solution metals, metal oxides

(conc.)(12 M). Evolves and carbonates

irritating fumes and must metal ores.

be handled in fume hood.

A strong and dangerous acid

Sulfuric Concentrated water Some organic sample

Acid H2SO4 solution is 96% H2SO4. dissolution (e.g., Kjeldahl­

A dense, syrupy liquid. Also aluminum and titanium

Reacts on contact with oxides

skin and clothing.

Evolves much heat when

mixed with water.

Nitric Available commercially Dissolving more noble

Acid, HN03 70% water solution (con- metals (e.g., copper and

centrated HN03). Reacts silver and some organic

with clothing and skin- samples.

turns skin yellow. Evolves

thick white and brown

fumes when in contact

with most metals.

Hydrofluoric Concentrated HF is a 50% Dissolving silica-

acid, HF water solution. Must be based materials and

stored in plastic contain- stainless steel.

ers, since it dissolves glass.

Very damaging to skin.

Perchloric Commercially available Dissolving difficult

Acid, HC1O4 as a 72% solution. organic samples and

stable metal alloys.

Aqua Ragia A 1:3 (by volume) mixture Dissolving highly

of concentrated HN03 and unreactive metals such as

concentrated HCI. gold.

Organic Mostly clear colorless Dissolving nonpolar

Solvents liquids that have high samples, or samples

vapor pressures and are requiring selective dis­

mostly nonpolar. Examples solution by extraction.

are hexane, ether, carbon

tetrachloride, and

methylene chloride

Grade classification of chemicals

• Practical or technical grade: 70-95% purity. Seldom used in the analysis; mostly used for cleaning

• Reagent grade: they meat the minimum required specifications of the Chemical Committee of the American Chemical Society. They possess high purity and may be used in quantitative analysis if the level of certain impurities does not affect the results

• Primary standard grade: exceptionally high purity. Impurities are determined using accurate instrumental methods. May be called AnalR in the UK.

• USP grade: used in the United States Pharmacopoeia. May be used in pharmaceuticals and food additives. Mainly must not contain health hazard impurities but may contain other impurities.

• Specific use chemicals: For water determination must be anhydrous, for HPLC or for spectroscopic measurenments

Handling Reagents

• Make sure that the reagent is suitable to the job

• Note any special warning on the label: poison, flammable, etc

• The impurity report fixed on the bottle stays as is whenever the purity is assured?

• Remember the following never:

– Never have the bottle open longer than is necessary to remove the desired portion of reagent

– Never return unused reagent to the bottle

– Never insert spoons, knives, or spatulas into a reagent bottle unless they are known to be clean and totally uncreative toward the reagent

• Remember the following never: In removing the reagent pour the desired quantity into a suitable container or use a clean porcelain or stainless steel spatula

• Try to avoid spillage of chemicals. If it happens clean them immediately specially when spillage takes place on the pans pf the balance

Safety

• Majority of safe lab practices are considered as a matter of common sense

• You should think and anticipate what will happen rather than just following the procedure blindly

• Use rubber gloves or wash your hands immediately when using toxic chemicals

• Hear are few of the more important do’s and don'ts

in the lab

- Know the location of all fire extinguishers,

showers, and eye wash fountains and how to use them.

- Label all chemicals and solutions that are stored outside

their original containers

- Keep work area clean

- Never begin work without knowledge of what you are doing

- Never bring, food or drinks into the laboratory

- Never work without a lab coat and never wear open toed or

open-top shoes

- Never work without approved eye protection

- Never heat flammable liquids over an open flame or outside a hood

Sample disposal

• In today's world, there is no place for the old sample disposal systems of pouring samples down the sink or dumping them in trash

• Lab supervisor must make sample disposal a priority among his duties

• Store the used samples in a separate place

• Develop environmentally safe disposal procedures

Glass-ware cleaning facilities

• If possible, a separate glass-ware facility should be established in the lab

• This will minimize a common source of contamination • Some labs found that high temperature baking of glassware

can significantly reduce blank values in trace organic analysis • Personnel working with this facility may require special

training • For quality assurance purposes, the training should be

documented.

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Semimicro and micro balances

• The macro or analytical balances perform weighing to the nearest 0.1 mg and loads up to 100 or 200 g

• The semimicrobalance is sensitive up to 0.01 mg (1 µg) and the microbalance is sensitive to about 0.001 mg.

• Grate care must be taken in their use.

• You should zero the balance whenever used. Zero point is not constant.

Fig. 2.1. Electronic analytical balance.

Modern balances are electronic. They still compare one mass against another since

they are calibrated with a known mass. Common balances are sensitive to 0.1 mg.

©Gary Christian,

Analytical Chemistry,

6th Ed. (Wiley)

25

Sources of Error in Weighing

• zero point drift

• Change in ambient temperature or

temperature of the object being weighed is

probably the biggest source of error, causing

a drift in the zero or rest point due to air

current convections.

• Hot or cold objects must be brought to

ambient temperature before being weighed.

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General Rules for Weighing

• Never handle objects to be weighed with the fingers. 'A piece of clean paper Learn these rules! or tongs should be used.

• Weigh at room temperature, and thereby avoid air convection currents.

• Never place chemicals directly on the pan, but weigh them in a vessel (weighing bottle, weighing dish) or on powder paper. Always brush spilled chemicals off immediately with a soft brush.

• Always close the balance case door before making the weighing. Air currents will cause the balance to be unsteady.

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Weighing of Solids

• Solid materials are usually weighed and dried in a

weighing bottle.

• These bottles have standard tapered ground-glass joints,

and hygroscopic samples (which take on water from the

air) can be weighed with the bottle kept tightly capped

• Weighing could be conducted by difference if the sample

is affected by atmosphere.

Fig. 2.7. Weighing dish.

A weighing dish or boat is used for direct weighing of samples.

©Gary Christian, Analytical Chemistry, 6th Ed. (Wiley)

29

Weighing of Liquids

• Weighing of liquids is usually done by direct weighing.

• The liquid is transferred to a weighed vessel (e.g., a weighing bottle), which is capped to prevent evaporation during weighing, and is then weighed.

• If a liquid sample is weighed by difference

by pipeting out an aliquot from the weighing bottle, the inside of the pipet must be rinsed several times after transferring.

• Care should be taken not to lose any sample from the tip of the pipet during transfer.

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Types of weighing

• There are two types: rough and accurate.

• Rough weightings to two or three significant figures are normally used when the amount of substance to be weighed need only be known to within a few percent.

• Examples are reagents to be dissolved and standardized later against a known standard

• Rough weighing should be done on a top load balance

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• Accurate weighings are reserved for obtaining the weight of a sample to be analyzed, the weight of the dried product in gravimetric procedures, or the weight of a dried reagent being used as a standard in a determination

• Accurate weight must generally be known to four significant figures to be used in calculating the analytical result.

• These are performed on an analytical balance, usually to the nearest 0.1 mg.

• An exact predetermined amount of reagent is rarely weighed (e.g., 0.5000 g), but rather an approximate amount (about 0.5 g) is weighed accurately (e.g., to give 0.5129 g).

• Some chemicals are never weighed on an analytical balance. Sodium hydroxide pellets, for example, are so hygroscopic that they continually absorb moisture.

• The weight of a given amount of sodium hydroxide is not reproducible (and its purity is not known).

• To obtain a solution of known sodium hydroxide concentration, the sodium hydroxide is weighed on a rough balance and dissolved, and the solution is standardized against a standard acid solution.

32

Volumetric Glassware

• The measurement of volumes of solutions is a very important measurement in titrimetric analysis.

• The volume of the titrant is used directly in the titrimetric % constituent calculation.

• Volumes of solutions are important measurements when preparing solutions for titrimetric analysis, whether such preparation is made by dilution or by weighing a pure solid chemical.

• A variety of reasons exist for transferring an accurately measured volume of a solution from one vessel to another. It is of utmost concern.

• There are basically three types of volume measuring devices in common use. These are

– volumetric flasks

– Pipet,

– buret.

33

Containers used for preparing solutions

Erlenmeyer flask Graduated cylinder Volumetric

Flask Beaker

N

o

Not accurate

34

Volumetric Flasks

• One important clue as to the accuracy of a volume

measured in any piece of glassware is the

narrowness of the tube on which the calibration line

is imprinted. The narrower this diameter, the more

accurate the measurement

• Solution volume must be brought up to the final

resting point of the bottom of the meniscus-i.e.,

bringing it up to the desired calibration line during

the solution

Fig. 2.8. Volumetric flask.

Volumetric flasks are calibrated

to contain an accurate volume.

See the inside back cover of the

text for tolerances of Class A

volumetric glassware.

©Gary Christian, Analytical

Chemistry, 6th Ed. (Wiley)

36

• Volumetric flasks are used in the diluting samples or solution to a certain volume.

• They come in a variety of sizes, from 1 L or more to 1 mL.

• These flasks are designed to contain an accurate volume at the specified temperature (20 or 25°C)

• The volume is reached when the bottom of the meniscus (the concave curvature of the upper surface of water in a column) just touches the etched "fill" line across the neck of the glass.

• just one drop (0.05 mL) of solution will make a visible change. That's an accuracy of ± 0.01%!

• The coefficient of expansion of glass is small, and for ambient temperature fluctuations the volume can be considered constant.

• These flasks are marked with "TC" to indicate "to contain." Other, less accurate containers, such as graduated cylinders, are also marked "TC.

37

• Volumetric flasks should never be used to transfer, or deliver, a specified volume.

• All pieces of glassware when used to deliver a volume of liquid have a thin film of the liquid remaining behind on the walls of the container.

• Some other glassware may be marked with “TD”

that is “to deliver”

38

• One problem with the volumetric flask exists because of its unique shape, and that is the difficulty in making prepared solutions homogeneous.

• A good mixing practice is to invert and shake at least a dozen times to ensure homogeneity.

• Additional accuracy is achieved with the use of class A flasks. Greater pains are taken at the factory, when calibrating these flasks, and they are thus considered to be more accurate.

• Also, Class A flasks always utilize a tapered ground glass or plastic cap, while others may utilize a plastic snap cap.

• The technician should always be aware of the type and size of cap required for a given volumetric flask.

• If any amount of solution should leak out due to an improperly sealed cap while shaking before the solution is homogeneous, its concentration cannot be trusted.

39

Pipets

• The pipet is used to transfer a particular volume of solution.

• It is often used to deliver a certain fraction (aliquot) of a solution.

• Usually, it has letters "TD" imprinted on it. It is usually calibrated to deliver the specified volume.

• ](The word "usually" is used here because there are two styles of pipets that are calibrated to contain and thus have the letters "TC" imprinted on them.)

• Pipets come in a variety of sizes and shapes.

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Volumetric or Transfer Pipet

41

Volumetric or Transfer Pipet

• The volumetric pipet is used for accurate

measurements, since it is designed to deliver only one

volume and is calibrated at that volume.

• Accuracy to four significant figures is generally

achieved, although with proper calibration, five figures

may be obtained if necessary.

• Most volumetric pipets are calibrated to deliver with a

certain small volume remaining in the tip.

• Under no circumstances should the last drop in a volumetric pipet be blown out with the bulb. The

volumetric pipet is not calibrated for "blow-out.“

• You should never suck solution by mouth but always Use a suction bulb to fill the pipet

42

43

• Class A volumetric pipets have a certain time in seconds imprinted near the top which is the time that should be allowed to elapse from the time the finger is released until the pipet is given the half-turn and removed.

• The reason for this is the film of solution adhering to the inner walls will continue to slowly run down with time, and the length of time one waits to terminate the delivery

• The intent with class A pipets, then, is to take into account this "run down" time by terminating the delivery by touching the the wall of the receiving container

44

• Measuring pipets are straight-bore pipets that are marked at different volume intervals.

• These are not as accurate, because nonuniformity of the internal diameter of the device will have a relatively larger effect on total volume than is the case for pipets with a bulb shape.

• Also, the drainage film will vary with the volume delivered.

• At best, accuracy to three significant figures can be expected from these pipets, unless you make the effort to calibrate the pipet for a given volume delivered.

Measuring Pipets

45

Measuring Pipets

Measuring pipets could be

Mohr pippets and Serological

pipets

calibration line stop short of the tip

calibration line go all the way

To the tip

46

47

Pipettors (Syringe pipets with disposable tips)

• They are in common use, mostly for pipeting extremely

small volumes. Some designs are shown below.

• These are generally considered to be precise, but not

necessarily accurate.

48

• These syringe pipets contains a disposable nonwetting plastic tip (e.g., polypropylene) to reduce both film error and contamination.

• A thumb button operates a spring-loaded plunger, which stops at an intake or a discharge stop; the latter stop is beyond the former to ensure complete delivery.

• The sample never contacts the plunger and is contained entirely in the plastic tip. These pipets are available in volumes of 1 to 1000 µL and are reproducible to 1 to 2% better.

• The actual volume delivered by these and other micropipets frequently does not need to be known because they are used in relative measurements. For example, the same pipet may be used to deliver a sample and an equal volume of a standard solution for calibrating the instrument used for the measurement.

• Precision in delivery is usually more important than the absolute volume delivered.

49

Syringe Pipets

50

Syringe Pipets

• They are used for both macro and micro volume measurements.

• The calibration marks on the syringes may not be very accurate, but the reproducibility can be excellent if an automatic deliverer is used, such as a spring load device that draws the plunger up to the same preset level each time.

• The volume delivered in this manner is free from drainage errors, because the solution is forced out by the plunger.

• .Microliter syringe pipets are used for introduction of samples into gas chromatographs.

• They are fitted with a needle tip, and the tolerances are as good as those found for other micropipets.

• The above syringe pipets are useful for accurate delivery of viscous solutions or volatile solvents; with these materials the drainage film would be a problem in conventional pipets.

• Syringe pipets are well suited to rapid delivery, and also for thorough mixing of the delivered solution with another solution as a result of the rapid delivery.

Fig. 2.13. Typical buret.

A 50-mL buret is marked in 0.1 mL increments.

You interpolate to 0.01 mL, good to about ±0.02 mL.

Two readings are taken for every volume measurement.

©Gary Christian, Analytical Chemistry, 6th Ed. (Wiley)

The Buret

52

The Buret

• A buret is used for the accurate delivery of a variable amount of solution.

• The conventional buret for macro titrations is marked in 0.1-mL increments from 0 to 50 mL

• The volume delivered can be read to the nearest 0.01 mL by interpolation (good to about ±0.02 or ±0.03 mL).

• Burets are also obtainable in 10-, 25-, and 100-mL capacities

• Microburets are available in capacities of down to 2 mL, where the volume is marked in 0.01-mL increments and can be estimated to the nearest 0.001 mL.

• Ultramicroburets of 0.1-mL capacity graduated in 0.001-mL (1-µL) intervals are used for microliter titrations.

53

• The meniscus illuminator has a white and a black field,

and the black field is positioned just below the

meniscus. Avoid parallax error by making the reading at

eye levyour eye level

54

General Tips for Accurate and Precise Titrating

• Before using a buret, you may have to grease

the stopcock.

• A thin layer of stopcock grease (not silicone lubricant) is applied uniformly to the stopcock, using very little near the hole and taking care not to get any grease in the hole.

• The buret is filled above the zero mark and the stopcock is opened to fill the tip.

• Check the tip for air bubbles. If any are present, they may work out of the tip during the titration

• Work air bubbles out by rapid squirting of titrant through the tip or tapping the tip while solution is flowing.

55

• The initial reading of the buret is taken by allowing it to drain slowly to the zero mark.

• Wait a few seconds to make certain the drainage film has caught up to the meniscus.

• Read the buret to the nearest 0.02 mL (for a 50-mL buret).

• The initial reading may be 0.00 mL or greater.

• The reading is best taken by placing your finger just in back of the meniscus or by using a meniscus

56

• The titration is performed with the sample solution in an Erlenmeyer flask. The flask is placed on a white background, and the buret tip is positioned in the neck of the flask. The flask is swirled with the right hand while the stopcock is manip­ulated with the left hand

• This grip on the buret maintains a slight inward pressure on the stopcock to ensure that leakage will not occur. The solution can be more efficiently stirred by means of a magnetic stirrer and stirring bar.

Proper technique for titration

Fig. 2.15. Proper technique for titration.

Place the flask on a white background.

Place the buret tip in the neck of the flask while your swirl.

©Gary Christian,

Analytical Chemistry,

6th Ed. (Wiley)

58

Care and Use of Volumetric Glassware

• If films of dirt or grease are present, liquids will not drain uniformly and will leave water breaks or droplets on the walls. Under such conditions the calibration will be in error.

• Initial cleaning should be with hot, dilute detergent, about 2%.

• A commercial laboratory soap called Alconox is often used

• Alcoholic KOH (KOH in ethanol) is very effective in dissolving greasy films

• Use of a buret or test tube brush aids the cleaning of burets and necks of volumetric flasks-but be careful of scratching the interior walls.

• Pipets should be rotated to coat the entire surface with detergent.

• Small volumetric flasks can be filled with the cleaning solution, but larger ones may be partially filled and tilted and rotated to coat the entire surface.

59

• Pipets and burets should be inverted and filled by suction (not by mouth!); a clamped rubber tube on the end will hold the solution in. Warm cleaning solution should remain in contact with the glass for about 15 minutes.

• Cleaning solution may be used at room temperature but is then slower acting and may have to be left in contact overnight.

• After any cleaning, the volumetric apparatus must be rinsed thoroughly with tap water and finally with several small portions of distilled or deionized water.

• Pipets and burets should be rinsed at least twice with the solution with which they are to be filled. If they are wet, they should be rinsed first with water, if they have not been already, and then a minimum of three times with the solution to be used; about one-fifth the volume of the pipet or buret is adequate for each rinsing.

60

• Note that analytical glassware should not be

subjected to the common practice employed in

organic chemistry laboratories of drying either in an

oven (this can affect the volume of calibrated

glassware) or by drying with a towel or by rinsing

with a volatile organic solvent like acetone (which

can cause contamination).

• The glassware usually does not have to be dried.

The preferred procedure is to rinse it with the

solution that will fill it.

61

Desiccators

• A desiccator is used to keep samples dry while they are cooling and before they are weighed and, in some cases, to dry the wet sample.

• Dried or ignited samples and vessels are cooled in the desiccator.

• A desiccator is an airtight container that maintains an atmosphere of low humidity.

• A desiccant such as calcium chloride is placed in the bottom to absorb the moisture.

• This desiccant will have to be changed periodically as it becomes "spent." It will usually become wet in appearance or caked from the moisture when it is time to be changed.

• A porcelain plate is usually placed in the desiccator to support weighing bottles, crucibles, and other vessels.

• An airtight seal is made by application of stopcock grease to the ground-glass rim on the top of the des-

Fig. 2.16. Desiccator and desiccator plate.

Use a desiccator to cool a dried or ignited sample.

Cool a red hot vessel before placing in the desiccator.

Do not stopper a hot weighing bottlle (creates a partial vacuum on cooling).

©Gary Christian, Analytical Chemistry, 6th Ed. (Wiley)

63

• A vacuum desiccator a side arm on the top for

evacuation so that the contents can be kept in a vacuum

rather than just an atmosphere of dry air. CaCl2 is

commonly used.