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The Analysis of Real Samples

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Steps of quantitative analysis1) Selecting a method2) Sampling3) Preparing a laboratory sample4) Defining replicate samples by mass or

volume measurements5) Preparing solutions of the samples6) Eliminating interferences7) Performing measurements for analyte

concentration8) Computing the results and estimating their

reliability

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Choice of analytical method1) Definition of the Problem

• What is the concentration range of the analyte to be determined?

• What degree of accuracy is desired?• What other components are present in the

sample?• What are the physical and chemical properties

of the gross sample?• How many samples will be analyzed?

2) Investigating the Literature• Chemical abstract/SciFinder• Web of Science

3) Testing the Procedure• The Analysis of Standard Samples• Using Other Methods• Standard Addition to the Sample

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Contents in sampling plan– Physically removing the sample from its

target population– Preserving and handling the sample– Storing the sample– Preparing the sample for analysis

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For Liquid Samples• Examples: beverages, urine, natural waters• Sampling tools: pipet, syringe, water sampler etc.• Preservation:

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For Gas Samples• Sampling tools: stainless steel canister,

Tedlar/Teflon bag, solid sorbent trap, filtering, cryogenic trap etc.

• For analyte removing:– Thermal desorption– Extracting with solvent

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For Solid Samples• Sampling tools: Grab sampler( 抓取式採樣器 )

、 Corer( 鑽取式採樣器 ) 、 Scoops( 杓 )/shovel( 鏟 ) 、 Thief ( 套管式採樣刀 )

• Sample preservation:– Low temperatures– Zero headspace– Adding Inert gas– etc.

• Sample preparation:– Reducing the particle size (to reduce

sampling variance)– Reducing the sample size (quantity)– Bringing solid samples into solution

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Bringing Solid Samples into Solution– Swirling and heating/Conventional

digestion/Wet digestion– Flux/Dry ashing– Microwave digestion

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Classifying Separation Techniques

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1. Filtration• Gravity/Suction/Pressure• Porous filter• Dissolved phase/particulate phase• Gravimetric analysis

Separations Based on Size

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2. DialysisA method of separation that uses a semipermeable membrane.

* Frequently used to purify proteins, hormones, and enzymes.

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3. Size-exclusion chromatography:• Also called gel permeation or molecular exclusion

chromatography• A separation method in which a mixture passes through a bed

of porous particles, with smaller particles taking longer to pass through the bed due to their ability to move into the porous structure.

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• If there is a difference in the mass or density of the analyte and interferent, then a separation can use centrifugation.

• The sample, as a suspension, is placed in a centrifuge tube and spun at a high angular velocity (high numbers of revolutions per minute, rpm).

• Particles of equal density, heavier particles having greater sedimentation rates.

• Particles are of equal mass, the highest density have the greatest sedimentation rate.

Separations Based on Mass or Density

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Example 1: Separated lysosomes from other components

1. Destroying the cell membranes.2. Centrifuge at 15,000xg (15,000 times the Earth’s gravity) 20

min.3. Isolate supernatant from the by decanting4. Centrifuge the supernatant at 30,000xg for 30 min.5. Leaving a residue of lysosomes.

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1. Establish the density gradients, e.g., solutions

of CsCl. (density gradient 1.65 g/cm3 ~ 1.80

g/cm3)

2. Place the the sample, e.g., mixture of proteins,

RNA, and DNA, into the centrifuge tube.

Protein

DNA

RNA

3. After centrifugation.

Proteins, with a density of less than 1.3 g/cm3 experience no sedimentation.

DNA, a density of approximately 1.7 g/cm3 separates as a band near the middle of the centrifuge tube.

RNA, with a density of greater than 1.8 g/cm3 collects as a residue at the bottom of the centrifuge tube.

Example 2: Equilibrium–density–gradient centrifugation

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Masking:A pseudo-separation method in which a species is prevented from participating in a chemical reaction by binding it with amasking agent to an unreactive complex.

Separations Based on Complexation Reactions (Masking)

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Cyanide is an appropriate masking agent for Ni2+ because the formation constant for Ni(CN)4

2– is greater than that for the Ni–EDTA complex.

Ni(CN)42– is relatively inert in the presence of EDTA.

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Changes in Physical State:

1. Fractional distillation

Boiling points versus composition diagramfor a near-ideal solution.

When the analyte and interferent are miscible liquids, for example, a low-boiling point analyte and a high-boiling point interferent.*The lower boiling point, the higher equilibrium vapor pressure.

Separations Based on a Change of State

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Equipment for a fractional distillation.

Temp.

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Changes in Physical State:

2. Recrystallization (fractional crystallization)

The solid sample is dissolved solvent, then cool down to promote the growth of large, pure crystal. The purified sample is isolated by filtration. Sample is dried to remove any remaining traces of the solvent.Additional recrystallizations if necessary.

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Changes in Chemical State:

1. Evaporation

SiO2 4HF SiF4 + 2H2OSiO2

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Other types ofChanges in Chemical State:

2. Selective precipitation

5. pH dependent precipitation

6. Complexation/extraction

7. pH dependent complexation/extraction

3. Electrodeposition

4. Ion exchange

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For a selective partitioning of the analyte or interferent between two immiscible phases. a phase containing a solute is brought into contact with a second phase, the solute partitions itself between the two phases:

Separations Based on a Partitioning Between Phases

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1. Liquid–Liquid Extractions:

The density of the two immiscible liquids determines which phase is the upper phase.For aqueous-organic extractions:

Density Lower Density Higher than H2O than H2O

Diethyl ether ChloroformHexane DichloromethaneToluene

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2. Solid-Phase Extractions:

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3. Solid-phase microextration (SPME):

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4. Continuous Extractions: (Soxhlet extractor for example)

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5. Microwave extractions:

The sample is placed in a sealed digestion vessel along with the liquid extraction phase, and a microwave oven is used to heat the extraction mixture. The extraction to take place at a higher temperature and pressure, thereby reducing the amount of time needed for a quantitative extraction.

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6. Purge and trap:

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7. Supercritical fluids extractions (SFE):

Supercritical fluid is a state of matter where a substance is held at a temperature and pressure that exceeds its critical temperature and pressure

For example, CO2 at 340 atm and 80oC has the properties between those of a gas and a liquid.

Density of supercritical fluid is high than that of gas, allowing good extraction ability from sample.

Viscosity of a supercritical fluid is significantly less than that of a liquid solvent, allowing it to pass more readily through particulate samples.

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End of Chapter 35-38