basic lab skills

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Title: Basic Laboratory Skills Training

1. Theory Basic Chemistry Overview

Chemistry is important to the patients who consume the products we produce in a number ofdifferent aspects. Chemistry is the basis behind drug research and development. New andinnovative products are discovered by combining certain molecules and chemicals andstudying the effects of these compounds on animals and humans. Once new drugsubstances have been discovered, the substance must be put into a form that is easilyconsumable and most effective. Whether the drug substance is to be put into solid dose,

aerosol, injection, dry powder, or topical form, studies must be done to show that the drugsubstance does not adversely interact with the inert ingredients. After all of the studies arecompleted and the drug substance is in a form ready for the consumer, the drug substanceand drug product must be tested to ensure that it is going to be safe and effective for thepatient.

Basic laboratory skills training ensures that the analyst will have the knowledge of basicprocedures and techniques to use in the quality control testing of drug substance andfinished products that will ultimately be consumed by the patient. Quality control testingultimately ensures the products we make and market are safe, effective and of high quality.

Chemistry is the study of matter. Matteris anything that has mass and takes up space andcan be visible or invisible. The physical and chemical properties of matter can bemeasured. Physical properties can be measured and observed without changing thecomposition of the substance. Some examples are density, specific gravity, boiling point,and appearance. How a substance behaves when it is around other substances isdescribed as chemical properties. For example, iron rusting in the presence of oxygenand water is a chemical property of iron.

Chemistry also deals with the properties and chemical reactions of the elements and theircompounds and how compounds can be formed. An element is a pure substance that iscomposed of only one kind of atom. Each element has a unique symbol associated with it.

A symbol can be one letter or two, but no more. The first letter is always capital and if there

is a second, it is always in lower case. Some of the common elements and their respectivesymbols are in the Table 1

Table 1 Common Elements and Symbols

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Aldebeiky Pharma (dBK)


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Element Name Element Symbol Element Name Element Symbol

Carbon C Hydrogen H

Oxygen O Nitrogen N

Calcium Ca Chlorine Cl

Iron Fe Magnesium Mg

Sulfur S Sodium Na

Potassium K Phosphorus P

An atom is the smallest particle of an element that retains the chemical properties of thatelement. All atoms of one element are the same, but atoms of one element are differentfrom atoms of a different element. Therefore, elements are unique because of the atomicstructure of atoms. Atoms are made of 3 different particles; protons, neutrons, andelectrons. Protons are positively charged particles, neutrons are neutral, or have nocharge, and electrons are negatively charged particles. The protons and neutrons form thecenter, ornucleus, of the atom while the electrons circle the nucleus in what is known as

an electron cloud. In a normal state, atoms are neutral because the number of protonsand electrons within the atom are equal and therefore each charge is cancelled out. If anelectron is lost or gained, this neutral state changes because the number of protons isimbalanced. If an electron is lost, there are more protons than electrons and therefore theatom has a positive charge. If an electron is gained, there are more electrons than protonsand the atom is considered negatively charged. An atom with a charge, whether positive ornegative, is called an ion. Each atom of each element has a different number of protonsand electrons and that is why atoms of different elements are different.

When two or more different elements are chemically combined in definite proportions, theresult is a compound. The atoms of the elements join to form compounds by gaining,losing, sharing, or trading electrons between one another. Unlike atoms and elements,compounds have different properties than the elements that make them up. For example,hydrogen and oxygen, two gases at room temperature, combine to make water, a liquid atroom temperature. Also, sodium, a metal at room temperature, combines with chlorine, apoisonous gas, to form table salt. The smallest unit of a compound that retains the chemicalcharacteristics of the compound is a molecule. Just as element names can be written as asymbol, compound names can be written as a combination of letters. For example, sodiumand chlorine combine to form sodium chloride, or table salt, and can be written as NaCl.This means that 1 sodium (Na) atom and 1 chlorine (Cl) atom combine to form a molecule ofNaCl. Water, H2O, is made of 2 hydrogen atoms and 1 oxygen atom. The number of atoms

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that combine does make a big difference. For example, CO2 is carbon dioxide, while CO iscarbon monoxide, a very deadly gas. Some common compounds and formulas are listedbelow in Table 2. You will come into contact with many of these and it will be very importantto be able to recognize them and associate them with their hazards.

Table 2 Common Compounds and Formulas

Compound Formula Compound Formula

Sodium Hydroxide NaOHMethyl Cyanide

(Acetonitrile)ACN

Methyl Alcohol(Methanol)

MeOH Water H2O

Sulfuric Acid H2SO4 Hydrogen Peroxide H2O2

Hydrochloric Acid HCl Phosphoric Acid H3PO4

SodiumBicarbonate

NaHCO3 Nitric Acid HNO3

Chemical compounds can be broken into different groups or families. They are divided intofamilies according to their behavior. The chemical families that will be most important toyour job are acids, bases, and organics.

Acids are compounds that have a pH of less than 7 and will turn litmus paper red. Acidsare corrosive, can cause chemical burns, and usually give off noxious fumes. Acids aregenerally very reactive and mixing an acid with water will usually produce a large amount ofheat. The chemical reactivity, not concentration, of the acid determines if the acid is

classified as being strong or weak. A strong acid is more reactive and hazardous than aweak acid at the same concentration. Some common strong acids are hydrochloric acid(HCl), sulfuric acid (H2SO4), and nitric acid (HNO3). Some common weak acids are citricacid (found in citrus fruits) and acetic acid (found in vinegar). There may be an occasionwhen you may be requested to make large volumes of a dilute acid from a concentratedacid. When making an acid, always add acid to water; never add water to a concentratedacid. If water is added to an acid, there is a possibility that the acid could react violently andspatter out of the container. When an acid is mixed with water, the atoms of the acid breakapart. For example, when HCl is added to water, the H and Cl atoms break apart and formH+ and Cl- ions. Acids have a large amount of H+ (protons) present.

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Bases are compounds that have a pH greater than 7 and will turn litmus paper blue. Likeacids, bases are corrosive, can cause chemical burns, and produce noxious fumes. Basescan also react violently with other chemicals and mixing a concentrated base with water willgenerate heat. In concentrated forms, bases and acids can react violently. Just as thereare strong and weak acids, there are also strong and weak bases. Some commonexamples of a strong base are sodium hydroxide (NaOH), potassium hydroxide (KOH), andammonia (NH3). Some common weak bases are calcium hydroxide (Ca(OH)2), andmagnesium hydroxide (Mg(OH)2). When a base is mixed with water, the molecules of thebase break apart into ions. For example, when NaOH is added to water, the Na and OHbreak apart and form Na+ and OH- ions. Bases have a large amount of OH- (Hydroxyl ions)

present.

Organic compounds are compounds that contain carbon and their presence is verycommon in our everyday lives. Some organics that you may be familiar with are petroleumproducts such as gasoline, alcoholic beverages, and acetone (nail polish remover). Organiccompounds are important in manufacturing areas, including pharmaceuticals. The twogroups of organic compounds that are most common in the pharmaceutical industry arehydrocarbons and oxygen-containing compounds.

Hydrocarbons are organic compounds that contain only hydrogen and carbon. Becausehydrocarbons are only made of two different elements, it seems that they would be fairly

simple compounds to understand. However, the number of hydrogens and carbons presentand their arrangement determines the properties of that compound. For example, thecarbons and hydrogens of C5H12 can be arranged differently to result in three differentcompounds. Some examples of common hydrocarbons are methane, propane, acetylene,and benzene.

Oxygen-containing organic compounds contain hydrogen, carbon, and oxygen. Thisgroup can be further broken into alcohols, organic acids, ketones, esters, and ethers.

Alcohols include ethanol and methanol. Some common ketones include acetone (nail polishremover) and formaldehyde (a preservative for dissection specimens). Citric acid (found incitrus fruits), acetic acid (found in vinegar) and acetylsalicylic acid (found in aspirin) aresome examples of organic acids. When an organic acid and an alcohol combine, an ester isformed. Many esters have pleasing odors and are found in natural and artificial flavors.Finally, ethers have in the past been used as an anaesthetic. Presently, they are morecommonly used as a solvent for other organic compounds.

Some hydrocarbons, alcohols, ketones, and ethers are often referred to as solvents. Asolvent is any chemical in which a material, or solute, is dissolved to form a solution. Asolute is the substance being dissolved. A solution is a type of mixture in which onesubstance (the solute) is dissolved in another (the solvent) to form a homogeneous (thesame throughout) mixture. Salt water is an example of a solution; oil and vinegar do not

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form a solution. Solutions can be liquid, gaseous, or solid. A substance is said to besoluble in another substance if it dissolves in that substance. Substances such as salt aresoluble in water, meaning the substances form a solution when combined. A substance isinsoluble if it does not form a solution (i.e. oil and vinegar). When as much solute isdissolved as possible, the solution is considered to be saturated. The amount of solute thatwill dissolve in a solvent depends on the chemical nature of the two and the conditions atwhich the substances are present. Temperature, pressure, pH, and mixing all affect thesolubility of a solute. In most cases, more solid solutes will dissolve at a higher temperature.Gaseous solutes usually dissolve better at a lower temperature and/or higher pressure. pHwill affect the solubility of some solutes and mixing generally increases solubility of a solute.

Another important term often used when referring to solutions is concentration.Concentration is defined as the amount of solute dissolved in a unit volume or weight of thesolvent. The concentration of a solution can be increased by increasing the amount ofsolute or decreased by decreasing the amount of solute. Concentration of a solute isusually defined as molar (M), normal (N), percent (% v/v, % w/w, or % w/v), or parts permillion (ppm).

Finally, as an analyst working in the laboratory, an understanding of acid base reactionsmay be helpful. Acid base reactions are only one type of many chemical reactions andmay be helpful in understanding the neutralization of acids or bases should you be required

to perform such a task. An acid base reaction is a reaction between a strong acid and astrong base that produces a salt and water (a neutral solution). A salt is any compoundwhose cation comes from a base and anion comes from an acid.

2. Laboratory Skills

2.1.Proper Handling of Glassware

- There are many types of specialized glassware used in the Chemistry laboratory. Eachtype has unique properties and qualities, and varying functions.

- Carefully inspect all glassware. Make sure that the glassware appears to be clean. Beforeusing the glassware, rinse it with a few milliliters of the sample solvent. If the glassware isdirty, return it to lab janitor for washing.

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- The glassware should not be chipped, cracked, broken, or excessively scratched. If theglassware appears to be broken or close to breaking, discard it in the dedicated containerfor broken glass.

- Always use the proper type of glassware for the correct purpose. For example, a beakershould never be used for accurate volumetric measurements.

2.2.Proper Use of Thermometers

Steps for use:

a) Choose a thermometer with appropriate range and graduations for the task you will bedoing.

b) Make sure that the thermometer is within the calibration date by checking the calibrationlabel affixed to the thermometer or its container.

c) Read a thermometer after a constant temperature has been attained. The correctionfactor should be added (whether positive or negative) to the temperature reading. Thisprovides the correct temperature.

2.3.Pipettes and Pipetting Techniques

- There are many types of pipettes. These include volumetric pipettes, micropipettes, andgraduated pipettes.

- Volumetric pipettes used in Laboratory Operations should be designated as class A, andare used for precise work. Volumetric pipettes have a single calibration line and have theletters "TD", meaning "to deliver", on them. Under no circumstances should the last drop ina TD volumetric pipette be blown out with the bulb into the receiving vessel.

Figure 2.3.1: The top of a class A pipette (positioned horizontally). Note the symbol "A"and the delivery time (35-sec.).

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Steps for correct use of pipettes: (Figure 2.3.2)

a) Ensure that the pipette is clean and the tip is not chipped.

b) Evacuate the pipette bulb (by squeezing).

c) Immerse the tip of the pipette into solution to be delivered.

d) Seat the bulb opening over the top opening of the pipette.

e) Hold the bulb in place while slowly releasing the pressure.

f) Continue to release the pressure while the solution is drawn into the pipette. (Re-evacuate the bulb if necessary.) Ensure that the tip of the pipette remains in thesolution at all times to prevent air bubbles from forming in the pipette. (Air bubblesdisplace solution which will result in a pipetting error.)

g) Draw the solution up well past the calibration line.

h) Quickly remove the bulb and seal the top of the pipette with a finger.

i) Keeping the finger in place, remove the tip from the solution and wipe the tip with atowel.

j) Slowly release the finger to adjust the meniscus (see Figure 2.3.1) to the calibrationline.

k) Place the tip over the receiving vessel and completely release the finger.

l) When the draining is complete, touch the tip to the inside wall of the vessel for about 2seconds.

Note: Never pipette by mouth.

Figure 2.3.2: Pipetting Technique

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Magnified tip view.


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After drainingtouch tip toglass to removethe last drop.

Dont blow out the remainingliquid. The pipet is calibratedfor this to remain.


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2.4. Preparing Volumetric Solutions

- Volumetric glassware is calibrated to contain a specified volume. The volumetric flasksused for analysis should be denoted as class A and should have "TC" meaning "tocontain" imprinted on it. Both of these symbols should be found on the base of the flask.

Dilution to the Mark (or qs meaning quantitate solution)

a) After transferring the solute, fill the flask about half-full with the sample solvent (the

diluting solvent), and swirl the contents to achieve solution.

b) Add more solvent and again swirl well. (Do not invert the flask.)

c) Bring the liquid level almost to the mark, and allow time for drainage. Allow the solutionto achieve room temperature. Then use a disposable transfer pipette to make such finaladditions of solvent as are necessary to bring the meniscus of the liquid to the mark.

d) Firmly stopper the flask, and invert repeatedly to assure uniform mixing.

e) If any amount of the solution should leak out due to an improperly sealed cap, whileshaking before the solution is homogeneous, its concentration cannot be trusted. The

solution should be discarded and a new solution prepared.

2.5. Solution Shelf Life

Consult the appropriate SOP for assigning expiration dates to laboratory prepared reagentsolutions. Reagent solution expiration dates must be checked prior to use. Reagentsolutions that have passed their expiration date must not be used for testing. Somesolutions may require special storage conditions, such as refrigeration.

2.6. Chemical Grades of Purity

Commercial or Technical Grade

This grade is used industrially, but is generally unsuitable for laboratory reagents because ofthe presence of many impurities.

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Practical Grade

This grade does contain impurities, but it is usually pure enough for most organicpreparations. It may contain some of the intermediates resulting from its preparation.

USP

USP grade chemicals are pure enough to pass certain tests prescribed in the United StatesPharmacopoeia and are acceptable for drug use, although there may be some impuritieswhich have not been tested for. This grade is generally acceptable for laboratory purposes.

Spectroscopic Grade

Solvents of special purity are required for spectrophotometry in the UV and IR ranges.Specifications of the highest order in terms of absorbance characteristics, water, andevaporation residues are given. The principal requirement of a solvent for these proceduresis that the background absorption be as low as possible. A label that states the minimumtransmission at given wavelengths accompanies most of these chemicals.

Chromatographic Grade (HPLC or GC)

These chemicals have a minimum purity level of 99+ mol % as determined by gaschromatography; each is accompanied by its own chromatogram indicating the column andparameters of the analysis. No individual impurity should exceed 0.2%.

Reagent Grade

Reagent grade chemicals are those which have been certified to contain impurities inconcentrations below the specifications of the Committee on Analytical Reagents of the

American Chemical Society (ACS). Each bottle is identified by batch number. Use onlyreagent grade in chemical analysis.

Primary Standard

Substances of this grade are sufficiently pure that they may serve as reference standards inanalytical procedures. You may use them directly to prepare standard solutions bydissolving massed amounts in solvents and then diluting them to known volumes. Primarystandards must satisfy extremely high requirements of purity; they usually contain less than0.05% impurities.

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2.7. pH

What is pH?

The term pH is derived from a combination of p for the word power and H for the symbolfor the element hydrogen. Mathematically, pH is the negative log of the activity of hydrogenions. However, many textbooks define pH as the negative log of the concentration ofhydrogen ions in solution (pH = -log[H3O+]). This definition is not truly accurate, because apH meter probe can not measure concentrations, it can only measure activity. Therefore,

the relationship illustrated in the following formula is the more accurate one:

pH = -log10a H+ wherepH represents the measure of the activity of hydrogen ions in asolution at a given temperature.

The term activity is used because pH reflects the amount of available hydrogen ions insolution instead of the concentration of hydrogen ions in solution.

In water or an aqueous solution, the following equilibrium exists between hydrogen ions(H+) and hydroxide ions (OH-):

H2O = H+ + OH-

The greater the number of hydrogen ions (H+), the more acidic the solution. The greater thenumber of hydronium ions (OH-), the more akaline or basic the solution.

The pH scale for aqueous solutions ranges from 0 to 14 pH units, with pH 7 being neutral.Water typically has a pH value of about 7. (Figure 2.7.1)

Figure 2.7.1: pH Scale

Although many of us are familiar with Litmus paper, the most accurate way to measure pH iswith potentiometric electrodes attached to a pH meter. These electrodes monitor the changesin voltage caused by differing concentration of hydrogen ions [H+]. These electrodes consist ofa pH sensing electrode and a reference electrode. The pH sensing electrode is only sensitive to

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hydrogen ions. The reference electrode provides a constant potential independent of theconcentration of hydrogen ions. The pH meter measures the potential between the pH sensingelectrode and the reference electrode. pH meters calculate results using the Nernst equation.

The Nernst Equation:

E = E0 + 2.303RT log a H+

nF

E = electrode potential

E0 = standard potential of the electrode

R = gas constant (8.31441 J K-1 mol-1)

T = temperature (in Kelvin)

n = valence (n = 1 for hydrogen ions)

F = Faradays constant

a H+

= activity of hydrogen ions

The following variables are determined prior to pH measurement.

The electrode potential (E) is measured by the electrodes.

The standard potential (E0) is determined by calibration of the electrode in a neutral solution

(pH = 7)

Temperature is measured by a temperature probe. If there is no temperature probe

connected to the pH meter, the temperature must be measured using a thermometer andentered into the pH meter.

The slope of the equation must be determined by calibration of the electrode using at least

two solutions of known concentration.

The slope equals the following part of the equation: 2.303RT logAH+

nF

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Note: Temperature is a critical variable in pH measurement. It affects the slope of theelectrode, sample and buffer pH values, and the potential of the electrode. Temperature mustalways be reported with pH.

The measurement of pH is one of the most common tests performed in laboratories. pHmeasurement is required for many pharmaceutical products before release to the market, due tothe affect of pH on the bio-availability of medications in the human body. It is used to verify thepH of the aqueous portion of HPLC mobile phases to ensure the quality of the chromotography.The pH of microbiological media must be tested to ensure that it is within a specific range for itto function properly. There are many laboratory uses for pH measurement.

pH Electrodes (figure 2.7.2)

In a discussion of pH, it is necessary to understand the electrode as the tool used to measurepH. pH electrodes can be either separate reference and sensing electrodes or combinationelectrodes. Combination electrodes are a combination of the reference and sensing electrodesin a single electrode. Combination electrodes are the most common type of electrode.Separate reference and sensing electrodes are normally only used for high precision researchapplications.

Figure 2.7.2: pH Electrodes

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pH Electrode Calibration

The most important step in accurate pH measurement is calibrating the pH electrode. (The pHmeter should be calibrated before use with the appropriate buffers to bracket the expectedsample range.) Calibration adjusts the slope and offset of the slope produced using the Nernstequation.

pH buffers are used as standards to calibrate a pH electrode. There are several commercialbuffers available, of different pH values. They should be traceable to national standards, local

standards of equivalent quality, or an absolute standard (e.g., a hydrogen electrode).Laboratory prepared buffers should be made from high quality reagents. The buffers below arerecognized by various regulatory bodies:

pH 1.68 at 25C

pH 4.01 at 25C

pH 6.86 at 25C

pH 7.00 at 25C

pH 9.18 at 25C

pH 10.01 at 25C

pH buffers are dependent on temperature. Many pH meters contain all of the temperatureprofiles for the buffers listed above. In this case, it is not necessary to adjust the profile.However, if the buffer is not one of the above standard buffers, then the pH meter profile mustbe adjusted. Otherwise, the pH meter will incorrectly adjust the calibration.

Buffers must be stored according to suppliers recommendations. Verify buffers are withinexpiration date before use. Do not re-use buffers and standards.

The standard procedure for calibration of a pH electrode is to select two buffers that bracket the

expected sample pH value. One of these buffers must be at pH 7.00.2 units. If there is arange of more than 4.0 pH units between these two buffers, a third buffer of intermediate pHmust also be used to check the calibration. If the sample pH does not lie between thecalibration points selected, the pH meter must be re-calibrated with suitable buffers.

Note: Consult the pH meter users guide for specific information on operation of your pH meter.

Sample Measurement

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a) Remove the electrode(s) from its storage solution and rinse it with distilled water. Dabthe electrode with tissue paper to remove excess water which could dilute the solution tobe tested. Do not wipe the glass bulb. Place the electrode and the temperature probeif applicable, into the first buffer to be tested. Wait for a stable response from theinstrument.

b) Calibrate the meter to read the temperature corrected value of the first buffer.

c) Rinse the electrode and probe and dab with a tissue to remove any excess water. Analternative rinsing procedure is to rinse with the next solution to be tested. Place the

probes in the next buffer. Wait for the reading.

d) Calibrate the meter to read the temperature corrected value of the second buffer.

e) Rinse the electrode as before. Place the electrode and temperature probe in yoursample and wait for a stable reading.

f) Ensure that the pH electrode bulb is immersed completely in the solution to bemeasured. Remove the electrode filling solution cover when measuring, to ensureproper flow of filling solution. The electrode should not touch the wall or the bottom ofthe sample container. The level of the sample solution should not be higher than thelevel of the filling solution. (Figure 2.7.3)

Figure 2.7.3: Electrode solution

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Solid samples should be dissolved in purified water. It is necessary that the water be carbondioxide free. The presence of dissolved carbon dioxide will cause significant bias in themeasure of samples with low buffering capacity.

It is difficult to measure the pH of solutions of very low conductivity due to the slow equilibrationof the electrode response. This problem can be alleviated by adding a drop of saturatedpotassium chloride solution to the sample to increase the conductivity.

Note: Consult the pH meter users guide and appropriate analytical method for specific

information on sample measurement.

Note: Stir all buffers and samples consistently. Choose one of the following methodsand use it for all measurements: A) stir continuously while taking measurements or B)stir, then stop stirring to take measurements. It is recommended to use a magneticstirrer at a moderate speed. Place some insulating material between your stir plate andyour sample container to prevent heating of your sample.

Electrode Maintenance

A regular maintenance schedule and proper storage of the pH electrode ensures properperformance, helps extend the life of the electrode, and avoids the cost of replacements.

On a weekly basis the electrodes should be inspected for scratches, cracks, build-up, andmembrane/junction deposits. The reference chamber of refillable electrodes should be drained,flushed with fresh filling solution and refilled. This maintenance procedure will keep theelectrode ready to use and improve the length of the life of the electrode.

An important part of maintenance is proper storage. Store the pH electrode properly. Chooseone of the following methods for storing the electrode: pH 7 buffer is good, pH 7 buffer withadded KCl is better, pH storage solution is best. Do not store a pH electrode in distilled water.

pH electrodes have a limited shelf life before use and should not be purchased and stored forlong periods.

2.8. Diluting Acids and Bases

Extreme care should be used when preparing acidic and basic solutions. All of thesesolutions should be prepared in the hood. When diluting a strong acid, the acid should beadded to water (NOT WATER TO STRONG ACID). Strong bases should be added towater. Be careful to wear the proper protective equipment when handling acids and bases.Do not inhale acids. Carefully rinse all glassware thoroughly that has had a concentratedacid or base in it, and dispose of acidic or basic waste in the proper waste can.

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2.9. Heating Solutions

Note: Obtain the proper equipment to handle the type of glassware that will be used.Tongs and oven mitts are available throughout the laboratory. Perform all heating ofsolutions in the hood when appropriate.

General Rules

a) Watch the solution closely.

b) Do not place hot glassware on a cold or wet surface; it may break because of thesudden temperature change.

c) Do not heat scratched or etched glassware.

d) Cool glassware slowly to prevent breakage.

e) When heating, allow for expansion (do not stopper a flask).

Steam Bath

Note: Use extreme caution when using steam.

a) Seat the flask on the steam bath after removing the proper number of supporting rings togive the maximum heating surface.

b) Perform steam bath experiments in the hood where appropriate.

c) When placing flasks on a steam bath, it might be necessary to place lead weights on theneck of the flask to keep the flask from overturning during the analysis.

Hot plates

a) Some hot plates have a built-in magnetic stirrer whose rotation speed can be adjusted.The rotating magnet underneath the glass container rotates the coated magnetic stirrerinside. This magnetic stirring bar is useful for heating and mixing operations that arecarried on simultaneously.

b) Use precautions when handling hot plates. The entire top surface heats and remainshot for some time after the hot plate has been turned off. It is preferable to leave a cardmarked "HOT" after use of the hot plate to let other analysts know that the hot plate hasrecently been used.

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c) Be sure that the electric cord, plug, and connector are in good condition. Do not use ahot plate with defective electric wiring or components.

d) Do not heat plastic coated laboratory glassware.

e) Never heat flammable solvents on hot plates.

2.10. Filtering Solutions

Mobile Phase

HPLC mobile phase is filtered utilizing a filtering apparatus and vacuum. Be sure to use flasksdesigned for vacuum filtration. Filters that should be used to filter mobile phase are 0.45 micron(unless indicated otherwise in the analytical method), and should be located in the approximatevicinity of the filtering apparatus. Also, confirm that the rubber stoppers located on the filteringapparatus fit snugly in the vacuum flask opening before starting filtration.

a) Connect the appropriate size of vacuum flask to the vacuum tubing.

a) Place the filtering apparatus into the opening on the vacuum flask. (Filters should beplaced in the apparatus shiny side down.)

b) Engage the vacuum by turning the knob all the way open.

c) Slowly pour the mobile phase into the filtering apparatus. Be careful not to overfill.Check for leaks.

d) After filtration is completed, disengage the vacuum and vacuum tubing from the flask.

e) Remove the filtering apparatus and carefully clean it by rinsing thoroughly with distilledwater.

f) Discard the filter paper in the trash after use.

Samples

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Samples may be filtered a variety of ways as there are many filter types available. These typesinclude; acrodisc, filter paper, and filter paper with filter holders. The analytical method shouldbe consulted about the proper validated filter type and pore size.

Unless otherwise stated in the analytical method, the first few milliliters of solution should bediscarded when filtering.

2.11. Using a Centrifuge

NOTE: Never open the cover on a rotating centrifuge.

Centrifuges rotate at high speed and effect separation by utilizing centrifugal force. The larger,heavier particles suspended in a liquid will be in the bottom of the centrifuge tube after anappropriate period of centrifugation.

a) Use an equal number of tubes or fill one with a counterbalancing solution of equalvolume (equal weight). This practice ensures that the centrifuge is properly balanced.Insert the tubes, equally spaced from each other. (The tubes should be arranged so thatthey are mirror images.)

b) Select the proper settings, per the analytical test method.

c) Close the cover and start the motor.

d) Wait until the centrifuge has stopped before opening the cover.

e) If an imbalance occurs, press the stop button, wait until the centrifuge has stopped, thenopen the cover and adjust to re-balance.

2.12. Weighing

Proper weighing (use of balances)

Each balance's linearity / calibration should be checked and recorded daily according to therelevant SOP.

The balance used for weighing analytical reference standards and other small amounts ofmaterial is the single pan, semi-micro balance (commonly called an analytical balance), with atotal capacity of 100 to 200 g and a sensitivity of 0.01 or 0.1 mg. The usual weighing operationconsists of first weighing a sheet of weighing paper or a receiving vessel on the balance pan.Ensure the doors or air shields on the balance are closed before taking a reading. Tare or zero

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the balance using the weighing paper or vessel. The substance to be weighed should be addedand a weight taken when a steady reading on the display is achieved. No chemical should everbe placed directly on the balance pan. This protects the balance from contamination andpermits complete recovery of the chemical being weighed.

Errors in weighing

Care should be exercised to minimize weighing errors. A vessel being weighed should not betouched with bare hands once tared, since fingerprints will change its mass. Never weighanything directly on the balance pan. A sample should always be at ambient temperature

before weighing to avoid errors due to convective air currents. Care should be taken to avoidcreating static charges while weighing. Ensure that the balance pan is clean prior to use.Residual substances could contribute to weighing error. A balance should be level prior to use.Many balances have a level indicator or bubble meter. A level balance is indicated by thebubble being positioned completely inside the bubble meter circle.

Sensitive balances are commonly placed on a marble slab to minimize the effect of roomvibrations on the reading. Adjustable feet and a bubble meter at the top allow the balance to bemaintained in a level position. A balance should be zeroed before use and between subsequentweighings.

Another type of balance located in the lab is a single pan top-loading balance used to weigh

large objects that do not require the sensitivity of a single pan balance or objects that weighmore than the limit of the single pan balance. The top loading balance is also used to weighlarger quantities of dry chemicals used for buffer solution preparation, mobile phase preparation,etc.

Weighing of analytical reference standards

a) Consult the analytical test method for all standards that are required to be prepared.

b) Retrieve these standards from the appropriate refrigerator.

c) Allow the standards to come to room temperature before weighing for at least one half

hour after removing from the refrigerator. Do not apply direct heat. The standard vialsmay be placed in a dessiccator during this period of time to minimize development ofcondensation in the vials. Use of a dessiccator is particularly important with hygroscopicmaterials.

d) Prior to opening the standard vial, tap gently to ensure the contents are at the bottom ofthe container. Always use a crimp cap remover to remove crimp caps to prevent injury.

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e) Care should be taken during transfer of the material from the vial to the weighingcontainer. Never return material to a standard vial once it has been removed, since thispractice may result in contamination of the stock standard.

f) Once a reference standard vial is emptied, place the empty vial in the bin specified forempty reference vial disposal.

2.13. Quantitative Transfer

In order to quantitatively transfer a solid, thoroughly rinse any items used during the transferwith the sample solvent into the receiving vessel.

When quantitatively transferring a liquid, thoroughly rinse the original container using the samplesolvent and add the rinse to the receiving vessel.

2.14. Dissolving Techniques

Manual shaking

a) When preparing to manually shake, the flask should be diluted almost to volume.

b) The flask should then be swirled to affect dissolution.

NOTE: Do not stopper and invert the flask until after you have diluted to the mark.

c) Fill to the volumetric mark with solvent, stopper and invert the flask numerous times.

NOTE: When inverting, make sure to completely fill the neck of the flask with thesolution.

Wrist action shakers

a) Wrist action shakers are typically used to shake vessels or aerosol cans for a definedperiod of time.

b) Carefully place the holders of the wrist action shaker around the vessel.

c) Test to ensure that the object is secure in the holder.

d) Set the shaking speed and time to shake per analytical test method (to start the shaker).

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Orbital shakers

a) Orbital shakers swirl the solution.

b) The flasks are placed firmly into the sample holders.

c) Set the orbiting speed, and turn the shaker on.

Vortex mixers

a) Vortex mixers create a rapid swirling action.

b) Typically, the vortex mixer is activated by placing pressure onto the top of the mixer.

c) Firmly hold the container while vortexing.

Sonication

Ultrasonic waves are used for cleaning glassware, mixing, dispersion, degassing, anddissolution of substances. Ultrasonic units use sound waves or mechanical vibrations that areabove the human hearing range. The sound waves are generated by the transducer thatchanges electrical energy into high frequency mechanical energy. This mechanical energy or

vibration is then coupled with the liquid in the tank. The vibration cause alternating high and lowpressure waves in the liquid. This action forms millions of microscopic bubbles that expandduring the low-pressure wave and form small cavities. During the high pressure waves thesecavities collapse or implode creating a mechanical action that dissolves solids.

Prolonged exposure to sonic waves will cause the solution to heat up. This may affect solutionstability and volume. Solutions should be allowed to cool to room temperature before diluting tovolume.

General tips:

a) The tank should be filled with at least 2 inches of water over the vessel holder screen.

b) A certain amount of heat is generated during the sonicating process. Do not becomealarmed if the bottom area of the bath becomes warm.

c) Provide adequate ventilation to the bath.

2.15. Proper Use of Mortar and Pestle

These laboratory tools come in a variety of sizes and shapes and are commonly constructed ofglass, porcelain, agate, mullite, and other hard materials.

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Steps for use:

a) Always clean the mortar and pestle thoroughly both before and after grinding eachsample.

b) Never grind two materials together unless specifically told to do so.

c) Place the substance to be ground into the mortar.

d) Carefully use the pestle to crush the substance.

e) Grind to a fine powder.

2.16. Titrating/Use of Burettes

Burettes work like measuring pipettes. Before being placed in service, a burette must bescrupulously cleaned. In addition, it must be established that the stopcock is liquid-tight.

Filling the Burette

Make certain that the stopcock is closed. Add 5 to 10 mL of solution and carefully rotate the

burette to wet the walls completely. Drain out through the tip. Repeat this procedure two moretimes. Then fill the burette above the zero mark. Free the tip of air bubbles by rapidly rotatingthe stopcock and allowing small quantities of solution to pass. Also check stopcock and burettefor air bubbles, and remove if necessary. Finally, lower the level of the solution to, or somewhatbelow, the zero mark. After allowing about a minute for drainage, take an initial volume reading.

Holding the stopcock

Always push the plug into the barrel while rotating the plug during a titration. A right-handedperson points the handle of the stopcock to the right, operates the plug with the left hand andgrasps the stopcock from the left side.

Performing a titration

a) Fill the burette as described in Filling the Burette section above.

b) Add the titrant to the titration flask slowly, swirling the flask manually, or with the use of astirring bar, until the end point is obtained. Keep the tip well within the titration vessel whileintroducing solution from the burette in increments of a milliliter or so. Swirl (or stir) the

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sample constantly to assure efficient mixing. Reduce the volume of the additions as thetitration progresses; in the immediate vicinity of the end point, the reagent should be addeda drop at a time. When it is judged that only a few more drops are needed, rinse down thewalls of the titration vessel (typically with water). Allow about a minute to elapse betweenthe last addition of reagent and the reading of the burette. Keep your eye level with themeniscus for all readings.

c) Near the end point, the trail of color from each drop is quite long. The end point is reachedwhen the color change does not disappear after 30 seconds.

d) Read the final position of the meniscus.

e) The difference between the "before" and "after" readings on the burette is the volume ofliquid delivered.

f) Never allow reagents to remain in burettes overnight. The stopcock may "freeze" becauseof prolonged contact with the reagents in the burette. Also, the concentration of the titrantmay change due to exposure to the environment.

2.17. Extraction Theory

- A solute may be soluble in different solvents that are immiscible with each other. For example:Bromine is somewhat soluble in water (4-g/100 mL) and very soluble in chloroform. Yet waterand chloroform are immiscible. When a solution of that solute in one of the two immisciblesolvents is shaken vigorously with the other immiscible solvent, the solute will be distributedbetween the two solvents in such a manner that the ratio of the concentrations (in moles perliter) of the solute is constant. (When a solution of bromine in water is shaken with chloroform,some of the bromine will transfer from the water to the chloroform. Since it is much moresoluble in the chloroform, the moles per liter concentration of it in the chloroform will be greaterthan the moles per liter concentration of it remaining in the water. However, the ratio of thesetwo concentrations will be constant; i.e. the same, regardless of how much water and chloroformis used.) This ratio is called the distribution coefficient, and it is independent of the volumes of

the two solvents and the total concentration of the solute.- This type of extraction transfers a solute from one solvent to another; i.e. transfer the brominefrom the water to the chloroform. It can be used to separate reaction products from reactantsand to separate desired substances from others in solution. The separatory funnel is used forthis purpose. Immiscible solvents, which are incapable of mixing with each other and willseparate from each other into separate phases, must be used. Miscible solvents, which arecapable of being mixed in any ratio without separation, can not be used.

- Multiple extractions with smaller portions of the extraction solvent are more effective than oneextraction with a large volume. The choice of extraction solvent determines whether the solute

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remains in the separatory funnel or is in the solvent that is drawn off. The solvent that has thegreater density will be the bottom layer. Thus the less dense solvent remains in the separatoryfunnel, and the more dense solvent is drawn off.

Use of Separatory Funnels

a) Use a clean separatory funnel.

b) Rinse the separatory funnel with an appropriate solvent, place the stopcock in thefunnel, and check for leaks.

c) Pour the solution to be extracted into the funnel, which should be large enough to hold atleast twice the total volume of the solution and the extraction solvent.

d) Pour in the extraction solvent. Close with the stopper.

e) Shake the funnel gently (or as specified in the analytical test method).

f) Invert the funnel and open the stopcock slowly to relieve the pressure built up.

Note: While opening the stopcock, point the open end away from yourself.

g) Close the stopcock while the funnel is inverted, and shake again.

h) Repeat steps e), f), and g), as needed to relieve pressure through out the extraction, butespecially in the beginning.

i) Place the funnel in a support and allow the two layers of liquid to separate. Removethe stopper closure.

j) Open the stopcock slowly and drain off the bottom layer.

k) Repeat operation, starting at step d), with fresh extraction solvent as many times as

required by the analytical test method.

l) Combine the lower layers that have been drawn off.

CAUTION: Always be certain that the stopcock is in a closed position before thefunnel is returned to the normal vertical position and that it is securely seated.

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2.18. Important Statistical terms

Mean

In the case in which a given measurement on a sample is repeated a number of times, theaverage of all measurements is called the mean. It is calculated by adding together thenumerical value of all measurements and dividing this sum by the number of measurements.

Standard Deviation

An overall picture of the quality of data (in terms of precision) is the Standard Deviation:

dsd + d + d + ...

n -1

12

22

32

=

in which ds represents the standard deviation. The significance of ds is that the smaller it isnumerically, the more precise the data and thus presumably the more accurate the data.

The standard deviation is used in most statistical methods in one way or another in evaluating

reliability, that is, in establishing the "confidence limits" for arriving at the final answer in theanalysis.

Relative Standard Deviation (%RSD)

In this case, ds is divided by the mean:

RSD =ds

m

Percent Relative Standard Deviation (%RSD) is calculated by multiplying the RSD by 100.

2.19. Good Documentation Practices

- Proper documentation is a very important part of quality control testing. Documentationshould be accurate and complete. It is the analysts responsibility to make sure that his orher work is accurate and complete. This saves time, confusion and cost.

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- Mistakes occur in various forms, the most common of which are write-overs, misspelledwords, and incorrect entries. While the person making the mistake is most likely to find andcorrect his own mistake, it is the responsibility of everyone who is accountable for thedocument to review for and correct errors.

- The recommended procedure for correcting these types of mistakes is to:

Use a single line to cross out the mistake. Do not obliterate, use whiteout, erase, orremove information.

Ensure that the original entry is still legible.

Make the correction.

Write a short explanation, if warranted.

Initial and date entry.

- N/A (Not Applicable) is used to omit a step or a portion of an operation from the document.The use of N/A requires an initial, date and sometimes an explanation.

- If a large amount of information needs to be omitted, the section should be boxed out, a

line drawn through it and N/A indicated. Boxing of steps or information lines are used toclarify or highlight the exact steps/information to be included as N/A. The use of boxing alsoensures clarity and legibility and avoids having to N/A a large number of steps.

- Every space in worksheet or batch assessment record should be filled. If the space is to befilled with data that is not applicable for the test done, a N/A is written. In other words, nospace on any document should be left blank.

- Comment Sections that are pre-printed on a batch or laboratory records MUST becompleted. If no comments are necessary, then it should be stated as such. The recordshould indicate "No Comments" or N/A in the section. When comments are entered theyshould be initialled and dated.

- All records should be legible and written in black, non-erasable ink.

- There may be times when the original page of a document becomes difficult to understanddue to too many cross outs, write-overs, or illegible writing. When this occurs a new pageshould be obtained from the Documentation Control Department. Photocopies are notacceptable. Accurate entries from the original are then transcribed onto the new page. Thenew page must be identified as a "REWRITE", attached to original and included in the batchrecord.

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- All instrument outputs (e.g. balance charts, chromatograms ...etc) should be attached,signed, and dated.

- All data located in another location should be clearly cross-referenced. (e.g. supportivedata or original plots)

- If a calculator was used to calculate any results; then a calculator tape must be attached tothe report and including the following data: the test performed, Products data (name, Lotnumber and other specific details) and finally the analysts signature and date.

chem / abdelkareem alazazzy

13/11/2008


basic lab skills

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