1. ERT207 ANALYTICAL CHEMISTRY
CHAPTER 3 : Preparing Samples for Analysis
Pn Syazni Zainul Kamal

2. CO2 : Ability to classify and use separation techniques and gravimetric methods for mass determination

3. What is Analytical Chemistry?
Concerned with the chemical characterization of matter and the answer of :
1) What is it (Qualitative) –identification of elements, ions or compound
2) How much is it (Quantitative)
“everything is made of chemicals. Analytical chemists determine what and how much”

4. Separation process
almost every element or compound naturally found in an impure state
these impure raw materials must be separated into their purified components before they can be put to productive use
a separation process is used to transform a mixture of substances into two or more distinct products.

5. Important in synthesis, chemical industries, biomedical
The separated products could differ in chemical or physical properties, such as size, or crystal modification or other separation into different components
The mixture at hand could exist as a combination of any two or more states: solid-solid, solid-liquid, solid-gas, liquid-liquid, liquid-gas, gas-gas, solid-liquid-gas mixture, etc.

6. Petroleum refinery plant
1st step – petroleum separation into fraction on the
basis of boiling point

7. Separation isolate the analyte from interfering constituents
Interferent – chemical sp. that causes a systematic error in an analysis
Separation isolate the analyte from interfering constituents
Separation can be either complete or partial
Complete separation
A B C
Mixture ABC
Partial separation
Mixture AB C
Mixture ABC

8. Classifying separation techniques
Separation based on ….
Size – filtration, dialysis, ion-exchange chromatography
Mass/density – (if there is diff. in the mass/density) centrifuge
Complexation reaction – masking

9. Change of state – (if an analyte and interferent in the same phase) – distillation, sublimation, recrystallization, precipitation
Partitioning between phase – (analyte and interferent are in two immiscible phases) – extraction, chromatography

10. Filtration – (size)

11. Centrifuge (mass/density)
Centrifugation is used when we want to separate small amounts of suspension.
The suspension of solid in liquid is poured into a centrifuge tube, then spin around very fast in a centrifuge. The spinning motion forces the solid to the bottom of the tube. Then the liquid can be poured off from the solid.
Centrifugation is commonly used in dairies to separate milk from cream to make skimmed milk. It is possible because milk has less density than cream.

13. Masking – (complexation reaction)
Used a masking agent which reacts with chemical species that may interfere in the analysis.
Masking agent react rapidly to form strong complexes with the ion to be masked

14. Sublimation – (change of state)
Some solids can change to vapor state without melting when heated. When the vapor is cooled, the solid forms again.
Eg iodine from a mixture of sand.
When heated, only iodine changes to vapor. The vapor changes back to solid on the side of the funnel.
Substances which sublime include anhydrous aluminum chloride, iodine and benzoic acid, anhydrous iron (III) chloride and anhydrous aluminum chloride.

16. Distillation – (change of state)
When a solution of solid in liquid is heated, the liquid will evaporates. The hot vapor that formed can be condensed back to liquid again on a cold surface.
A way is to recover water from a salt solution. The solution is heated and the stream is to be condensed back to water. The solute and solvent can both be collected.
Evaporation + Condensation = DISTILLATION

18. Separation based on partitioning between phase

19. 2 techniques can be use to separate analyte and interferent:
Extraction (is which a solute is transferred from one phase to a new phase)
Chromatography (will be discussed in chapter 6)

20. Type of extraction Liquid-liquid extraction Solid-phase extraction
Continuous extraction

21. Liquid-liquid extraction
Very useful for isolate analytes from complex sample matrices prior to chromatographic analysis and spectrophotometric determination.
Method to separate compounds based on their relative solubility in two different immiscible liquids. (water & solvent)
Liquid-liquid extraction involve distribution of a solute between two immiscible liquid phases
‘Solvent extraction’ – perfume, biodiesel, vege oil

22. Distribution coefficient
If a solute is in aqueous phase and is extracted into an organic phase:
A solute S will distribute itself between two phases (after shaking/mixing).
Ratio of [S] in the two phases will be constant.
KD – distribution coefficient
KD = [S] solvent 1 eg. organic solvent
[S] solvent 2 eg. water

23. Distribution process in liquid-liquid extraction
Using separatory funnel
The mixture is put into a separatory funnel,
The mixture is shaken for about a minute, and the phases are allowed to separate.
A solute is extracted from an aqueous solution into an immiscible organic solvent.
A solute is an organic compound, will distribute from water into organic solvents.
The principle is “like dissolves like”.
at bottom layer, the solvent is denser and will be drawn off after the separation is completed.

24. Liquid-liquid extraction

25. The distribution ratio
Many substances, partially ionized in aqueous layer as weak acid
Eg extraction of benzoic acid from an aqueous solution. Part of HBz in aqueous layer exist as Bz-.
HBz is a weak acid in water with ionization constant Ka
Distribution ratio: is the ratio of the concentrations of all the species of the solute in each phase.
D = [HBz]e
[HBz]a + [Bz-]a
Organic solvent
Aqueous solvent

26. Ka = [H+]a[Bz-]a
[HBz]a
Equation that relates D, KD and Ka is
D = KD
1 + Ka/[H+]a
the extraction efficiency will be independent of the original concentrations of the solute.

27. Percent extracted
Fraction of the solute extracted will depend on the volume ratio of the two solvents
Fraction of solute extracted is equal to milimoles of solute in the organic layer divided by the total number of milimoles of solute.
The milimoles are the molarity times the mililiters.
Therefore, the percent extracted is given by,
%E = [S]oVo
[S]oVo + [S]aVa
X 100%

28. Vo and Va are the volumes of the organic and aqueous phases, respectively.
Relation between %E and D is given by,
%E = D
D + (Va/Vo)
If Vo = Va, then
%E = 100D
D + 1
If D is less than 0.001, the solute can be considered quantitatively retained.
The percent extracted changes only from 99.5% to 99.9% when D is increased from 200 to 1000.

29. Example 1
20 ml aqueous solution of 0.10 M butyric acid is shaken with 10 ml ether. After the layers are separated, it is determined by titration that 0.5 mmol butyric acid remains in the aqueous layer. What is the distribution ratio, and what is the percent extracted?

30. Solution Starting in aqueous = 20 ml x 0.10 M BA = 2.0 mmol BA
Left in aqueous sol = 0.5 mmol BA
Extracted in ether = 1.5 mmol BA
[BA]e = 1.5 mmol/10ml = 0.15M
[BA]a = 0.5 mmol/20ml = 0.025M..therefore
D = = 6.0
0.025
%E = D = 100 x = 75%
D + (Va/Vo) (20/10)

31. Solid-phase extraction
Liquid-liquid extraction – extracting solvents used limits to those that are water immiscible, Large volume of solvents used – waste disposal problem
SPE – become widely used for sample cleanup prior chromatographic analysis
compounds that are dissolved or suspended in a liquid mixture are separated from other compounds in the mixture according to their physical and chemical properties.

32. SPE uses the affinity of solutes dissolved or suspended in a liquid (mobile phase) for a solid through which the sample is passed (stationary phase) to separate a mixture into desired and undesired components.
The result either - desired analytes of interest or undesired impurities in the sample are retained on the stationary phase.

33. The stationary phase comes in the form of a packed syringe-shaped cartridge
Solid phase extraction cartridges and disks are available with a variety of stationary phases, each of which can separate analytes according to different chemical properties.
Most stationary phases are based on silica that has been bonded to a specific functional group.

34. Some of these functional groups include hydrocarbon chains of variable length (for reversed phase SPE), quaternary ammonium or amino groups (for anion exchange), and sulfonic acid or carboxyl groups (for cation exchange)

36. Working principle Conditioning
Powdered phase (sorbent extraction bed) placed in a small cartridge
Powdered phase eg silica bonded with hydrophobic phase, must be conditioned in order to interact with aqueous sample
Accomplish by passing methanol through the sorbent bed.
This penetrate into bonded layer and permit water mol. and analyte to diffuse into bonded phase
Water is passed to remove excess solvent prior to adding sample

37. Loading sample/retention
The sample (dissolve in liquid) is passed through sorbent extraction bed to separate a mixture into desired and undesired components.
Analytes adsorbed to sorbent extraction bed
Rinsing/washing
Rinse the column to remove undesired components
Elution
Remove desired analytes

39. Continuous extraction
For component of interest that has unfavorable partition coefficient (desired compound insoluble in solvent).
Extraction is accomplished by continuously passing the extracting phase through the sample until a quantitative extraction is achieved
Involving solid samples are carried out with a Soxhlet extractor

41. Working principle
solid material containing some of the desired compound is placed inside a thimble made from thick filter paper, which is loaded into the main chamber of the Soxhlet extractor.
The Soxhlet extractor is placed onto a flask containing the extraction solvent.
The Soxhlet is then equipped with a condenser.

42. The solvent is heated to reflux.
The solvent vapour travels up a distillation arm, and floods into the chamber housing the thimble of solid.
The condenser ensures that any solvent vapour cools, and drips back down into the chamber housing the solid material.

43. The chamber containing the solid material slowly fills with warm solvent.
Some of the desired compound will then dissolve in the warm solvent.
When the Soxhlet chamber is almost full, the chamber is automatically emptied by a siphon side arm, with the solvent running back down to the distillation flask.
This cycle may be allowed to repeat many times, over hours or days.

44. After extraction the solvent is removed, typically by means of a rotary evaporator, yielding the extracted compound.
The non-soluble portion of the extracted solid remains in the thimble, and is usually discarded.