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Basic concepts and tools of
Analytical Chemistry
L3 2hr
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Outline
• SI units • Measuring weights and volumes • Preparing a solution • Analytical reagents, chemicals • pH determination • Buffer solutions • Data analysis
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SI: Units
• Science has units • SI: abbreviated from the French ( Système International d’unités)
– Derived from the Système métrique (metric system) – In 1790 the French Revolution implemented the metre and kilogram as standards of length
and mass
• Then later it was refined and extended to – Length : meter (m) – Mass: kilogram (kg) – Time : second (s) – Electric current: ampere (A) – Thermodynamic temperature: kelvin (K) – Amount of substance: mole (mol) – Luminous intensity: candela (cd)
• Only 3 countries have not officially adopted the metric system – Liberia, Birmanie, USA – (although in the US most scientific measurements are using the metric system)
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Non SI units accepted with SI
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• Sometimes SI units are not practical • Non SI units accepted with SI
– Minute, hour, day, hectare, litre, tonne, ..
• Non SI units whose values must be obtained – Electron volt, dalton, dalton/unified atomic mass unit, Planck
constant, electron mass, ..
• Other non-SI units – Bar, angström, nautical mile, decibel, ..
1 liter
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A word of English
• French : mètre, litre, gramme, kilogramme • British English: metre, litre, gramme, kilogramme • American English: meter, liter, gram, kilogram
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Expressions e.g. la7n: exempli gra+a, for example i.e. la7n: id est, in other words,
Concentrations units
• Concentration = mass / volume – e.g.
• gram per liter; g/L = g. L-1
• mg/L, ng/mL, µg/mL
• Molarity – 1 mole per liter = 1 mol/L = 1 mol.L-1 = 1M – 10-6 mol/L = 10-6 mol.L-1 = 1 µM
• Conversion µM to ng/mL – If the molecular weight of the compound is MW – then 1 µM = MW x ng/mL – e.g.
• MW of compound X is 300 then 1µM of compound X is 300 ng/mL
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1 mg/L = 1 µg/mL 1 µg/L = 1 ng/mL
Mass vs weight
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• Mass and weight are the same in common language
• But weight is a force
The pound 1 lb = 0.453 59237 kg
Measuring weights and volumes
• It is important to know the concentration – Concentration = mass/ volume
• We have have to measure with accuracy mass and volume
• A balance is an analytical instrument that measures weight – But it is calibrated with mass so it measures mass
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We need the right tool Weighing scales
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> 1000 kg 10-‐ 150 kg .1-‐5 kg
A balance has an useful range We have to choose the correct scale
∆ ∼ 50 g ∼ 200 g ∼ 100 kg
Analytical balance
• To weigh mg and g amounts we need an analytical balance
• It is an analytical instrument – It should be
• accurate • reproducible • sensitive
• Should be regularly calibrated with standard masses • Has a door to prevent air draft
– precision : ± 0.1 mg
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Measuring volumes
13 Accuracy and precision
We have to choose the correct container
How to read the volume?
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Laboratory pipettes
• Pasteur • Graduated (measuring) pipettes, burets • Volumetric • Mechanical electronic pipettes
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pipette
• Pasteur pipettes – Named after Louis Pasteur – in glass or in plastic to deliver small volumes (no accuracy
intended) – Cheap enough to be considered disposable
• But can be reused if no contamination possible
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Graduated and volumetric pipettes
• Graduated pipettes – To deliver known volumes – In glass or polystyrene for sterile serology
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burette
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• Graduated glass cylinder to dispense liquid drop by drop with accuracy
Volumetric pipettes
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Safety: Never pipet with your mouth
Mechanical/electronic pipettes
• Fixed volume or adjustable • Need a disposable plastic cone
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Small volume syringe
• For small volumes – In the µL range
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uncertainty
• No measurement is free from error
• Error is introduced by – The limitations of instruments and measuring devices – The imperfection of human senses
• In analytical chemistry the estimated degree of error in a measurement is called the uncertainty of the measurement and the reported values are only with significant figures
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Significant figures
• Numbers that describe the value without exaggerating its accuracy – We report as significant all numbers knows with aboslute
certainty, plus one more digit that is inderstood to contain some uncertainty
– The uncertainty in the final digit is usually assumed to be ± 1
• There are some rules – http://2012books.lardbucket.org/books/principles-of-general-
chemistry-v1.0m/s05-09-essential-skills-1.html
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Last significant figure in weighing
• The reading on the analytical balance is 3.2479 mg
• How good is that number ? – Should we report it ? – We know that the balance is accurate
• The precision of the balance is 0.1 mg. It means that it can detect a change of 0.1 mg
• The weight should be reported as 3.2 ± 0.1 mg – Why ? – The last significant number is the first decimal: .1 mg – The numbers 4 7 9 are meaningless. The balance cannot detect changes of:
• . 01, .001 and .0001 mg
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Significant figures in measuring volume
• Which measuring apparatus do you take to deliver 9.7 mL as accurately as possible ? – Then with how many significant figures can you measure the
volume ?
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Use the 10mL graduted cylinder Accurate with 2 significant figures
hVp://2012books.lardbucket.org/books/principles-‐of-‐general-‐chemistry-‐v1.0m/s05-‐09-‐essen7al-‐skills-‐1.html
Amount and concentration
• 1 mole of any substance is N molecules of that substance – Avogrado number N= 6.022 1023
• It is the number of carbon atoms in 12 g of 12C • N unit: mol-1
• In biochemistry we are usually calling the unified atomic mass unit a dalton Da (from John Dalton) – kDa kilodalton: for high mass polymers: proteins and nucleic acids
• The molecular weight of a compound is the sum of the masses of the elements – It is the sum of the mass of each constituent
– H2O = 2 H + O = Da – CH4 = C + 4 H = Da
• Molecular weight calculator – http://www.lenntech.com/calculators/molecular/molecular-weight-calculator.htm
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Molarity
• I mole of a molecule contains N molecules with a mass expressed in g (MW)
• 1 mole of water is 18.02 g of water • 1 mole of NaCl is 58.44 g of NaCl
• Conversely the Molecular Weight gives the number of g in a mole of substance – 18.02 g of water contains
• 1 mole of H2O molecules • N (Avogadro number) water molecules
• Concentration – mol/L = mol.L-1 = M
• 1M NaCl = 1 mol/L NaCl = 58.44 g/L • A 0.1M NaCl solution contains 5.844 g of NaCl per liter
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Prepararing a solution
• It is one of the most important task of bioanalytical chemistry
• What is a solution ? – A solution is a chemical compound (or chemicals) dissolved in a
liquid • usually at a defined concentration
• There are 3 components in a solution – The chemical(s) – The solvent – The concentration
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Chemicals
• Bionalytical chemistry is usually about measuring trace amounts/ concentration in a biological sample – so the chemicals should not add impurities in the system
• A chemical compound of a known high standard of purity has to be used (Analytical grade) – There are specifications for reference compounds – We have to know what are the impurties
• i.e. glucose
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Chemicals for analytical work
• References compounds must be as pure as possible • You have to know what is in the compounds
– Racemate/ enantiomer ? – % pure ? – What are the impurities ?
• An analytical chemical/reagent needs a Certicate of analysis
• You get the chemical that is not giving interference with your analysis
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American Chemical Society
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hVp://pubs.acs.org/reagents/demo/
solvent
• A solvent is a liquid chemical
• The solvent should not add impurities to the system
• Water is a solvent – Water has to be as pure as possible
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Storage of chemicals
• A chemical/reagent/solution has a finite shelf-life which depends on: – Temperature:
• At what temperature should I store the chemical? • Room temperature/ refrigerated/ frozen
– Humidity • Some compounds are hygroscopic • You need to store them in a dessicator
– Length of time • A chemical may degrade with time. It has a limit date
– Light • A chemical may be light sensitive
– Oxidation • A chemical may be subject to oxidation
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concentration
• The concentration may be expressed in 3 different ways: – Mole per Liter: M (or mM or µM, ..)
• mol/L – mass per volume g/L
• The isotonic NaCl 0.9 % is a – 0.9 g/100mL of water – 9 g/L
– Volume per volume : vol/vol • When we mix 2 solvents the solution is usually expressed in vol/vol • Ex: a 40% (vol/vol) methanol/water used in chromatography
• When we talk about a solution we must know – how it is prepared: M, mass/vol or vol/vol – and when it was prepared
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Preparing a solution
• To prepare 500mL of a 0.1 M NaCl solution – How much do we need ?
• 0.1 x 0.5 mole of NaCl – 0.1 x 0.5 mole of NaCl = 0.1 x 0.5 x MW(NaCl) in g – = 0.1 x 0.5 x 58.44 = 2.922 g of NaCl
– We dissolve 2.922 g in 500 mL
• We calculate the number of moles in the desired volume to get the right concentration
• The date of preparation should always be noted on the container
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Tricks
• Some compounds are present (crystallize) as: – Salts – Hydrates
• Potassium phosphate tribasic anhydrous K3PO4 • Potassium phosphate tribasic, octahydrate K3PO4, 8 H2O
• So when we prepare a solution we have to know the physical form – Anhydrous, Hydrate, Free base, salt –
• Ex trehalose is present – Anhydrous : melting point 203°C, molar mass: 342.296 g/mol – Dihydrate: melting point 97 °C, molar mass: 378.33 g/mol – If we want a tetralose 1M trehalose solution we disolve in 1 L
• 342.296 g if we start with the anhydrous form • 378.33 g if we start with the dihydrate
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Tricks: acidic and basic compounds
• With a basic compound always check: – Base form
• Morphine: C17H19NO3 MW= 285.34 – Salt and what kind of salt
• Morphine hydrochloride: C17H19NO3.HCl MW = 312.81 • Sulfate pentahydrate: 2C17H19NO3.H2SO4.5H2O
• With an acidic compound always check – Acid form – Salt and what kind of salt
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Dilution
• We have a solution and we want to dilute it: • 2 ways
– Final amount is the same • the final volume is increased
– Final volume is the same • The final amount is decreased
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Dilution Final amount is the same
• We have a 10M HCl solution and we want to prepare 200mL of a 0.5 M HCl solution – What volume should we take ?
– c1V1 = c2V2: the amount is the same before and after – C1 concentration before dilution – C2 concentration after dilution – V1 volume before dilution ( unknown) – V2 volume after dilution (what we want)
• V1 = (0.5/10) x 200 10 mL de HCl 10 M
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V1 = (c2/c1). V2
Dilution the amount does not change
• The amount does not change, the volume increases – 10 fold dilution
• 1 vol starting solution + 9 vol solvent – 20 fold dilution
• 1 vol starting solution + 19 vol solvent
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Serial dilution the volume does not change
• Serial dilution – Can be easily automated
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Beware: the error in pipe7ng propagates
V1 = (c2/c1). V2
errors
• We have seen that measurements are uncertain • volume 25 ± 0.03 mL • Weight 10 ± 0.1 mg
• Absolute uncertainty vs relative uncertainty – For the 25 mL volumetric pipette the volume is ± 0.03 mL
• 0.03/ 25 = 0.0012 = 0.12% • It is a fixed volume
– The balance weighs at ± 0.1 mg • If we weigh
– 1 mg there is 10% uncertainty – 10 mg there is a 1% uncertainty
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Error in concentration
• Concentration = mass / volume • The error (uncertainty) of the concentration is the sum of
the errors
• Ex: mass = 100 ± 1 (1%) Volume = 100 ± 1 (1%) • both values between 99 and 101
– The ratio may be between 99/101 and 101/99 • 0.98 and 102 = 100 ± 2% • (more on that later)
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∆ (concentra7on) = ∆(mass) + ∆(volume)
Measuring the pH of a solution
• With a pH meter • With pH indicators
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��� pH meter
• pH meter – Precise ± 0.1 or ± 0.01 pH unit – Measures the potential difference between the working electrode
(usually glass) and a reference electrode – There are many hand held pH-meters – It is calibrated against solutions of known pH values
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pH indicators
• It is a chemical that changes color – in a specific pH range
• It changes color in a pH interval
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Bromocresol green
phenophthalein
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pH = 1 pH = 13
pH indicators test papers
• The paper has been impregnated with dyes • The paper is dipped into the solution
– the color is compared to a standard chart
• Gives an indication
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Tutorials
• Mole – http://chemcollective.org/activities/tutorials/stoich/the_mole
• Dilution
• Virtual lab – Can be downloaded – Chemcollective.org/vlabs
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