Basic concepts and tools of

Analytical Chemistry

L3 2hr

1  

Outline

•  SI units •  Measuring weights and volumes •  Preparing a solution •  Analytical reagents, chemicals •  pH determination •  Buffer solutions •  Data analysis

2  

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)

3  

4  

Non SI units accepted with SI

5  

•  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  

6  

A word of English

•  French : mètre, litre, gramme, kilogramme •  British English: metre, litre, gramme, kilogramme •  American English: meter, liter, gram, kilogram

7  

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

8  

1  mg/L  =  1  µg/mL  1  µg/L    =  1  ng/mL  

Mass vs weight

9  

•  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

10  

We need the right tool Weighing scales

11  

>  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

12  

Measuring volumes

13  Accuracy  and  precision    

We  have  to  choose  the  correct  container  

How to read the volume?

14  

Laboratory pipettes

•  Pasteur •  Graduated (measuring) pipettes, burets •  Volumetric •  Mechanical electronic pipettes

15  

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

16  

Graduated and volumetric pipettes

•  Graduated pipettes –  To deliver known volumes –  In glass or polystyrene for sterile serology

17  

burette

18  

•  Graduated glass cylinder to dispense liquid drop by drop with accuracy

Volumetric pipettes

19  

Safety:  Never  pipet  with  your  mouth    

Mechanical/electronic pipettes

•  Fixed volume or adjustable •  Need a disposable plastic cone

20  

Small volume syringe

•  For small volumes –  In the µL range

21  

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

22  

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

23  

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

24  

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 ?

25  

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

26  

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

27  

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

28  

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

29  

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

30  

American Chemical Society

31  

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

32  

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

33  

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

34  

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

35  

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

36  

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

37  

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

38  

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

39  

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

40  

Serial dilution the volume does not change

•  Serial dilution –  Can be easily automated

41  

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

42  

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)

43  

∆  (concentra7on)    =  ∆(mass)    +  ∆(volume)      

Measuring the pH of a solution

•  With a pH meter •  With pH indicators

44  

��� 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

45  

pH indicators

•  It is a chemical that changes color –  in a specific pH range

•  It changes color in a pH interval

46  

Bromocresol  green  

phenophthalein

47  

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

48  

Tutorials

•  Mole –  http://chemcollective.org/activities/tutorials/stoich/the_mole

•  Dilution

•  Virtual lab –  Can be downloaded –  Chemcollective.org/vlabs

49