Chapter 30

Introduction to Separation

Dong-Sun Lee/ CAT-Lab / SWU 2010-Fall version

Petroleum refinery. The first step in the refining process is to separate petroleum into fractions on the basis of boiling point in large distillation towers.

The major separation procedure is distillation, in which the crude oil is passed through heaters where the temperature is raised to approximately 340 oC, at which temperature all of the gas, gasoline, jet fuel, and light fuel oil fractions are in the vapor phase. This vapor and liqid mix- ture enters a distillation tower.

Separations

Introduction

A sample that requires analysis is often a mixture of many components in a complex matrix. For samples containing unknown compounds, the components must be separated from each other so that each individual component can be identified by other analytical methods. The separation properties of the components in a mixture are constant under constant conditions, and therefore once determined they can be used to identify and quantify each of the components. Such procedures are typical in chromatographic and electrophoretic analytical separations.

Introductory documents on separation procedures

• Chromatography

• Centrifugation

• Electrophoresis

• Crystallization, Precipitation

• Distillation, Steam distillation, Reduced pressure distillation

• Evaporation

• Extraction

• Ultrafiltration

• Freeze drying, Freeze concentration

• Reverse osmosis

• Surface adsorption

• Gas stripping

Separation by precipitation

Precipitation methods are valuable for separating ions from one another. For this purpose it is not necessary to have a precipitate of exact stoichiometric composition suitable for weighing. After filtration and washing, the precipitate may be redissolved (usually by adding acid) and the isolated element(s) determined by titration or other means.

Examples:Separations based on control of aciditySulfide separationsSeparations by other inorganic precipitantsSeparations by organic precipitantsSeparation of species present in trace amounts by precipitationSeparation by electrolytic precipitationSalt-induced precipitation of proteins (Salting out)

Extraction of metal ions by dithizone into CCl4.

[S2– ] = 1.2 ×10–22 / [H3O+]2

Solubility of Sulfide (example : HgS )

Step 1. HgS = Hg2+ +S2– , S2– + H2O = HS– + OH– ,

HS– + H2O = H2S + OH– , H2O = H+ + OH–

Step 2. 2[Hg2+ ] + [H1+] = 2[S2–]+[HS–]+[OH–]

Step 3. [Hg2+ ] = [S2–]+[HS–]+[H2S]

Step 4. Ksp =[ Hg2+][S2–] = 5 ×10-54

Kb1 = [HS–][OH–] / [S2 –] = 0.80, Kb2 = [H2S][OH–] / [HS–]= 1.1 ×10-7,

Kw = [H+ ][OH–] = 1.0 ×10–14

Step 5. six equations, six unknowns

Step 6. If pH=8.00 [OH–] = 1.0 ×10–6 ,

[H2S]= Kb2 [H2S] / [OH–] = 0.11 [HS–]

[HS–]= Kb1 [S2 –] / [OH–] =8.0 ×105 [S2 –]

[Hg2+ ] = [S2–]+[HS–]+[H2S]

= [S2–]+ 8.0 ×105 [S2 –]+0.11 × 8.0 ×105 [S2 –]

=(8.88 ×105 [S2 –]

Ksp = [Hg2+][S2 –] = [Hg2+]{[Hg2+]/ (8.88 ×105 )}= 5 ×10-54

[Hg2+] = 2.1×10–24 M

Separation by Extraction

Introduction

Extractions use two immiscible phases to separate a solute from one phase into the other. The distribution of a solute between two phases is an equilibrium condition described by partition theory.

Boiling tea leaves in water extracts the tannins, theobromine, and caffeine (the good stuff) out of the leaves and into the water. More typical lab extractions are of organic compounds out of an aqueous phase and into an organic phase.

Analytical Extractions

Elemental analysis generally requires fairly simple (not necessarily easy) sample preparation. Solids are usually dissolved or digested in caustic solution and liquids are sometimes extracted to separate the analyte from interferences.

Organic analysis is often much more complicated. Real-world samples can be very complicated matrices that require careful extraction procedures to obtain the analyte(s) in a form that can be analyzed.

Examples of organic solvents used for solvent extraction include;

Chloroform is a heavy solvent which is widely used for extracting organic compounds and metal complexes from aqueous solution.

Benzene and ethyl ether are lighter than water solvents.

The degree of separation afforded by solvent extraction will be determined by the distribution ratio, that is the relative concentration of the solute in the organic and aqueous phases.

Separation can be improved by increasing the number of extractions, for instance three × 10 cm3 extractions will give a better separation than a single 30 cm3 extraction.

Solvent extraction

Extraction refers to the transfer of a solute from one liquid phase to another.

Solvents that are less dense than water :

Diethyl ether (d= 0.7077, 25oC)

Benzene (d= 0.8787, 15oC)

Hydrocarbons (ex. Hexane, d= 0.660, 20oC)

Solvent that are immiscible with and dense than water :

Chloroform (d= 1.484, 20oC)

Dichloromethane (d= 1.33479, 15oC)

Carbon tetrachloride (d= 1.589, 25oC)

Parts of a separatory funnel

Stopper

Body

Stopcock

Drain Tip

Aqueous layer

Organic layer

(ex. methylene chloride)

Liquid-Liquid extraction

(solvent of higher density than liquid being extracted)

Liquid-Liquid extraction

(solvent of lower density than liquid being extracted)

Solid-Liquid extraction

http://www.chemglass.com/

Simultaneous distillation and extraction (SDE), Soxhlet extraction

Liquid-Liquid extraction : The partition law.

Let's have an aqueous solution of trichloroacetic acid. Our need is to remove the acid from the solution.  This can be easily accomplished by mixing, in a separatory funnel, an organic solvent (e.g., dimethyl ether) immiscible with water but able to solubilize the acid. After mixing,  the system will reach a state of equilibrium which is governed by the partition law:

K = [Ce] / [Cr]

where K = partition or distribution coefficient

Ce = concentration in the extract phase

Cr = concentrations in the raffinate phase

K  = a constant (for ideal  solutions) at constant temperature which depends on the nature of solvents used.

Partition coefficient

S(in phase 1) S(in phase 2)

V1 V2

K = AS2 /AS1 [S]2 / [S]1

K = {(1–q)m/V2} / (qm / V1)

q = V1 / (V1 + KV2)

where q is the fraction of solute remaining in phase 1.

Multiple extractions

for n extractions

q n = [V1 / (V1 + KV2)] n

Partitioning of a solute between two liquid phase

Multiple extractions

From the definition of the partition coefficient we have that

K = [Ce]/[Cr]  , [Ce] = K[Cr]

and we get:

[Co] Vr = [Ce]Ve + [Cr]Vr   =  K[Cr]Ve + [Cr]Vr = [Cr] (KVe + Vr)

[Cr] = [Co] Vr / (KVe + Vr) = [Co] / (1 + KVe/Vr)

where Co is the initial concentration of the solute and

Vr the volume of the initial solution;

[Ce] is the concentration of solute in the extract phase and

Ve the volume of the extract phase;

[Cr] is the concentration of solute in raffinate phase and

Vr the volume of raffinate phase. Please note that the volume of the raffinate phase

(Vr) is supposed to be equal to that of the original solution.

for n extractions

[Cr]n = [Co] [ (1 + KVe/Vr)]n = [Co] [Vr / (KVe + Vr)]

n

Numerical example

The partition coefficient of a solute is 3, assuming that the initial solution contain 0.01 M of solute in 100 ml of solution, let's calculate the amount of solute (in % fraction) remaining in the raffinate after:   1) a single extraction using 500 ml of solvent E

2) five extraction using 100 ml of solvent E

1. In the first case   q = [100 / (100 + 3×500)] = 0.062 = 6.2 %

2 In the second case q = [100 / (100 + 3×100)]5 = 0.00098 = 0.098 %

Extraction of Metal - Organic Complexes

A large number of organic compounds form complexes with metal cations. Many of these complexes are more soluble in organic solvents than in water and hence are extractable. For example, the iron(III)-cupferron chelate or the aluminium(III)-oxine complex is extracted by chloroform. The distribution ratio for this type of complex is usually very large, so only one or two extractions of the complex is usually very large and so only one or two extractions with fresh solvents are needed. The chelating reagents are solids and the metal complexes are insoluble in water. The metal is extracted by adding an aqueous solution of the reagent to an aqueous solution of the sample and extracting the precipitated metal complex with an organic solvent. Alternatively, the chelating reagent may be dissolved in a water-immiscible organic solvent, which is then shaken with the sample solution. Shaking produces a temporary emulsion of the two liquid phases. Metal ions in the sample solution react with the chelating reagent at the interfaces between phases and then are extracted into the organic phase.

Extraction methods used in the making of aromatic materials.

Aromatic Materials of Natural Origin

Steam Distillation

ExpressionMaceration Digestion

PercolationEnfleurage SFE

Distilled essential oils

Citrus oils

Concretes

Pomades

Absolutes

Fat or

Fixed oil

Alcohol tincture

Solvent Extraction

Petroleum benzin

Alcohol

SFE Absolute

Hydrosol

Chemical Analysis & Technology Lab / SWU

Steam distillation for the extraction of essential oils

Chemical Analysis & Technology Lab / SWU

Major extraction techniques for solid, liquid and gaseous samples.

Fiber(HS, Direct immersion)

In tube

Solid Samples

Liquid Samples

Gaseous Samples

HS-HDME

LPME

SPTE

SPE

SPME

SFE

HS-SE

MBE

SBSE

Single drop hollow fiber

Stir bar

Liquid trapping

Purge & Trap

Soxhlet extraction

Solvent extraction

Tube Column switching

Disk cartridge

Well plate

SDME

Solid Phase Extraction (SPE)

Analytical scale SPTE & field work

Khs

•••

Klh

••••••

•

HS-HD-LPME

Solid phase trapping & solvent extraction(SPTE) device

SPME apparatus to collect fragrances.

Solid phase micro-extraction (SPME) fiber assembliesHub Color Stationary Phase Description

Red 100m Poly(dimethyl)siloxane non-bonded

Yellow 30 m Poly(dimethyl)siloxane non-bonded

White 85 m Polyacrylate partially crosslinked

Black 75m Carboxen-Poly(dimethyl)siloxane partially crosslinked

Gray 50/30 m StableFlex Divinylbenzene/ highly crosslinked

carboxen/PDMS

SUPELCO, Bellefonte, PA

Headspace SPME vs. Direct SPME

Needle

Headspace

Fiber

SS tubing

Separating Ions by Ion Exchange

Cation-exchange resins contain acidic groups, while anion-exchange resins have basic groups.

Strong-acid type exchangers have sulfonic acid(—SO–3H+) groups attached

to the polymeric matrix and have wider application than weak-acid type exchangers, which owe their action to carboxylic acid (—COOH) groups.

Similarly, strong-base anion exchangers contain quaternary amine

[—N(CH)+3OH –] groups, while weak base types contain secondary or

Tertiary amines.

x RSO–3H+ + Mx+ = (RSO–

3)xMx+ + x H+

Solid solution solid solution

x RN(CH)+3OH – + Ax– = [RN(CH)+

3]x Ax– + x OH–

Solid solution solid solution

Structure of a cross-linked polystyrene ion-exchange resin.

Schematic home water softener.

2 RSO–3H+ + Ca2+ = (RSO–

3)2Ca2+ + 2 H+

Solid solution solid solution

(RSO–3)xMx+ + x Na+ = x RSO–

3H+ + Mx+ (Regeneration)

Solid solution solid solution

Chromatography

Introduction

Chromatography is a separations method that relies on differences in partitioning behavior between a flowing mobile phase and a stationary phase to separate the the components in a mixture. A column (or other support for TLC, see below) holds the stationary phase and the mobile phase carries the sample through it. Sample components that partition strongly into the stationary phase spend a greater amount of time in the column and are separated from components that stay predominantly in the mobile phase and pass through the column faster.

As the components elute from the column they can be quantified by a detector and/or collected for further analysis. An analytical instrument can be combined with a separation method for on-line analysis. Examples of such "hyphenated techniques" include gas and liquid chromatography with mass spectrometry (GC-MS and LC-MS), Fourier-transform infrared spectroscopy (GC-FTIR), and diode-array UV-VIS absorption spectroscopy (HPLC-UV-VIS).

1. "Chromatography" is a named by and M.S. Tswett applied to a variety of techniques used for analytical separations. 1) "Chroma" is Greek for "color." 2) "Graphein" is Greek for "to write."

2. Originally, chromatography was used to separate plant pigments. "Colors" developed and separated as plant homogenates were passed through calcium carbonate-packed columns. 3. Common features of chromatographic methods include... 1) A stationary phase, often packed into a column through which the sample is passed to effect separation. Stationary phases can be almost anything. 2. A mobile phase, the liquid (or gas) that carries the sample/analyte through the stationary phase.4. Because different analytes have different binding affinities for the stationary phase, their movement through the stationary phase will be retarded by varying degrees, affecting separation.

Classification of chromatography

1) Column chromatography

GC ----- GSC…. Adsorption

GLC…. Partition

LC ------ LSC …. Adsorption

LLC …. Partition

IEC ….. Ion exchange

EC --- GPC ….Gel permeation

GFC ….. Gel filtration

AC ….. Affinity

2) Plane chromatography

Paper

Thin layer

Chromatography was invented by the Russian botanist Mikhail Tswett.

Classification of Chromatographic Methods

1. Planar chromatography 1) The stationary phase is supported by a flat surface (e.g., a glass plate, a plastic sheet, paper, etc.). 2) The mobile phase and analyte pass through the stationary phase by capillary action and/or gravity.

Thin-Layer Chromatography (TLC)

Introduction

Thin-layer chromatography (TLC) is a chromatographic technique that is useful for separating organic compounds. Because of the simplicity and rapidity of TLC, it is often used to monitor the progress of organic reactions and to check the purity of products.

Method

Thin-layer chromatography consists of a stationary phase immobilized on a glass or plastic plate, and an organic solvent. The sample, either liquid or dissolved in a volatile solvent, is deposited as a spot on the stationary phase. The constituents of a sample can be identified by simultaneously running standards with the unknown. The bottom edge of the plate is placed in a solvent reservoir, and the solvent moves up the plate by capillary action. When the solvent front reaches the other edge of the stationary phase, the plate is removed from the solvent reservoir. The separated spots are visualized with ultraviolet light or by placing the plate in iodine vapor. The different components in the mixture move up the plate at different rates due to differences in their partioning behavior between the mobile liquid phase and the stationary phase.

The AUTOSPOTTER is a semi-automated device used to apply samples on Thin Layer Chromatography plates. The instrument eliminates the need for manual sample application and can be used to apply up to 18 samples at a time. http://www.analtech.com/data_tlc.php3?ps_session=48ce73290363f5dba3971cf8aa9e1f21

Fig. Glass chromatography tank (30 cm x 10 cm x 25 cm high,pictured with Chrom Clips and compression rod) for developing paper chromatograms and TLC plates.http://www.lplc.com/misc/tlc.htm

Spotting a TLC plate with sample: Running the TLC plate in solvent.

http://www.chem.vt.edu/chem-ed/sep/tlc/tlc.html

http://www.alkemist.com/services.htm

Developing solvent

Origin

Spot A

Spot B

Solvent front

XA XB Y

Rf =distance from the origin migrated by a compound

distance from origin migrated by solvent

2. Column chromatography

The column is packed with a solid phase and equilibrated by passing the mobile phase through the column.

An aliquot of mobile phase containing dissolved analytes is applied to the top of the column and allowed to "drain" into the bed of the column.

Mobile phase is continuously fed into the top of the column to flush the analytes through.

As the analytes pass through the column, they "partition" between the mobile phase and stationary phase.

1. Analytes with lower affinity for the stationary phase spend less time bound to the column and pass through quickly. 2. Analytes with higher affinity for the stationary phase spend more time bound to the column and pass through more slowly.

Column chromatography

1) The stationary phase is packed into a glass column, tube or other cylindrical container. 2) Mobile phase and analyte are passed through the column under pressure or by gravity. Consider the liquid chromatography example shown below:

Schematic representation of a chromatographic separation

Concentration profiles of solute bands A and B at two different times in their migration down the column.

Two-component chromatogram illustrating two methods of improving separation:

(a) Original chromatogram with overlapping peaks

(b) Improvement brought about by an increase in band separation

(c) Improvemnet brought about by a decrease in the widths.

Description of Column Elution Chromatography

Terminology: 1. Eluent - mobile phase; solution fed into the top of a column to elute a sample. 2. Eluate - species eluted from the bottom of a chromatographic column.

eluent in --- column ---- eluate out

3. Chromatogram - a plot of the detector signal (or any other function of analyte concentration) versus elution time (or elution volume).

4. Chromatograph - device (instrument) that performs the chromatographic separation (e.g., the HPLC instrument).

5. Absolute retention time (tR) - time (from the moment of injection) required for a sample to reach the detector (or be eluted from a column).

6. Adjusted retention time (tR’) – additional time required for a sample solute to travel

the length of the column, beyond the time required by unretained mobile phase. tR

’ = tR – tm

7. Dead time (tm ) - time required for unretained mobile phase travels through the column. (Air is often injected on purpose as in internal reference.)

The Chromatogram : a plot of the detector response as function of time

Retention time : tR

Retention volume : VR = tR × flow rate Solvent peak Base line Peak height Peak area Peak size is proportional to the quantity of material passing through the system

Analysis of Chromatograms

A. Retention times of peaks may to useful for qualitative identification of specific species in mixtures. 1. Under a given set of conditions, a single analyte will always have the same retention time. 2. A control sample of the analyte is run to determine the retention time of the analyte, and the presence of the analyte in a complex, unknown sample is usually determined by the presence of a peak identical in retention time to the control sample.

3. It is always best to repeat the analysis with a different set of chromatographic conditions. If the control sample and complex, unknown sample have peaks that match under more than one set of chromatographic conditions, it is usually safe to assume that they are the same species.

B. Peak heights and areas may be used to derive quantitative data from chromatograms. 1. The concentration of an analyte is proportional to the area (or height) of the peak observed on the chromatogram. 2. To quantify an analyte, a series of control runs with varying amount of standard analyte must be run to prepare a standard curve (a plot of peak area versus amount of standard run). 3. The unknown sample is then run under conditions identical to the control runs, and the area (or height) of the analyte peak is determined. Area is usually determined by... a. Electronic integration of the peak. b. Approximation by triangulation.

c. Cutting out the peak with scissors from a paper copy of the chromatogram and weighing the paper peak. The weight is proportional to area.

Chromatogram

Retention parameters of chromatography

Capacity factor (= retention factor ; capacity ratio ; partion ratio )

k’ = ( tR – tm ) / tm

= tR’ / tm

= (VR – Vm ) / Vm

= (CsVs ) / (CmVm)

= (KVs) / Vm

Separation factor (= relative retention = selectivity factor)

= ( tR1 – tm ) / ( tR2 – tm )

= k2 / k1

Resolution of two peaks

Rs = ( tR1 – tR2 ) / wav

= tR / wav

= VR / wav

where is the separation between peaks (in units of time or volume) and wav

Is the average width of the two peaks in corresponding units.

Factors affecting resolution :

Rs = (N1/2 / 4){(–1)/}{k2’ / (1+kav’)}

Band broadening of chromatography

A band of solute broadens as it moves through a chromatography column. One main cause of band spread is diffusion.

Standard deviation of band : = (2Dt)1/2

where D is diffusion coefficient(m2/s) and t is time.

The flux of molecules diffusing across a plane of unit area is proportional to the concentration gradient and to the diffusion coefficient :

Flux(mol/(m2s) = J = – D (dc/dx)

Column efficiency :

Plate height (=height equivalent to a theoretical plate: HETP: H) is the constant of proportionality between the variance of the band and the distance it has traveled(x). The smaller the plate height, the narrower the band width.

2 = 2Dt =2D(x/v) = (2D / v)x = Hx

H = 2 /x

Where v is linear flow rate (distance / time).

The number of theoretical plates (N) in the entire column is the length(L) divided by the plate height(H).

= w/4 L = x

N = L / H = Lx / 2 = L2 /2 = 16 L2/w2

N = 16 tR2/w2 = (tR

2 / 2 ) = 5.55 tR2 / w½ 2

Asymmetric peak

N {41.7 (tR/w0.1)2 / (A/B + 1.25)

Plates in a fractionating column

van Deemter equation for plate height

H A + (B/v) + Cv

Where v is the linear flow rate and A, B, and C are constants.

A = multiple paths (eddy diffusion) term

(B/v) = longitudinal diffusion term

Cv = equilibration time term

In packed columns, all three terms contribute to band broadening.

For open tubular columns, multiple path term (A) is zero, so bandwidth decreases and resolution increases.

In capillary electrophoresis, both A and C go to zero, thereby reducing plate height to submicron values.

van Deemter graph

Effect of mobile phase flow rate on plate height for

(a) Liquid chromatography

(b) Gas chromatography

Band spreading from multiple flow paths.

Longitudinal diffusion Effect of equilibrating time

Isotherms and their resulting chromatographic band shapes.

dslee@swu.ac.kr

http://mail.swu.ac.kr/~cat