IF WE KNOW D, CAN WE FIND THE PERCENT RECOVERY?

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139



E≡



amt o

amt (total)



where amto refers to the amount of analyte extracted into the organic phase and

amt(total) refers to the total amount of analyte originally present in the aqueous

sample. The fraction extracted can be expressed as follows:

E=



C 0Vo

Dβ

=

C 0Vo + C aq Vaq 1 + Dβ



(3.16)



where β is defined as the ratio of the volume of the organic phase, Vo, to the volume

of the aqueous phase, Vaq, according to

β = Vo /Vaq

The percent recovery is obtained from the fraction extracted, E, according to

% Recovery or % E = E × 100

Equation (3.16) shows that the fraction extracted and hence the percent recovery

depend on two factors. The first is the magnitude of the distribution ratio, which is

dependent on the physical/chemical nature of each analyte and the chemical nature

of the extractant. The second factor is the phase ratio β. The magnitude is usually

fixed if the extractant is not changed, whereas the phase ratio can be varied. If,

instead of a single-batch LLE, a second and third successive LLE is carried out on

the same aqueous solution by removing the extractant and adding fresh solvent, the

%E can be maximized. After allowing time for partition equilibrium to be attained,

while keeping the phase ratio constant, it can be shown that a second successive

extraction will extract E(1 – E) while a third successive extraction will extract

E(1 – E)2. The fraction remaining in the aqueous phase following n successive LLEs

is (1 – E)n–1. To achieve at least a 99% recovery, Equation (3.16) suggests that the

product βD must be equal to or greater than 100. Even with a product βD = 10, two

successive LLEs will remove 99% of the amount of analyte originally in an aqueous

environmental sample.12



10. ARE ORGANICS THE ONLY ANALYTES THAT

WILL EXTRACT?

Our examples so far have focused on neutral organic molecules such as acetic acid.

The majority of priority pollutant organics of importance to TEQA are neutral

molecules in water whose pH values are within the 5 to 8 range. Before we leave

the principles that underlie LLE, the answer to the question just posed is yes.

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Consider the significant difference in KD for NaCl vs. HOAc partition constants

discussed earlier. Ionic compounds have little to no tendency to partition into a

moderate to nonpolar organic solvent. If, however, an ion can be converted to a

neutral molecule via chemical change, this ion can exhibit a favorable KD. This is

accomplished in two ways: chelation of metal ions and formation of ion pairs. The

mathematical development of a metal chelate is discussed in this section.

A number of organic chelating reagents exist that coordinate various metal ions,

and the metal chelate that results consists of neutral molecules. This neutral or uncharged

metal chelate will have a KD much greater than 1. Metal ions initially dissolved in

an aqueous phase such as a groundwater sample can be effectively removed by metal

chelation LLE. Commonly used chelating reagents include four-membered bidentate

organic compounds such as dialkyl dithiocarbamates, five-membered bidentates such

as 8-hydroxyquinoline and diphenyl thiocarbazone, dithizone, and polydentates

such as pyridylazonaphthol. 8-Hydroxyquinoline, commonly called oxine (HOx), is

the chelating reagent used in this section to introduce the mathematical relationships

for metal chelation LLE. Similar equations can be derived for other chelating

reagents.

Figure 3.6 depicts the principal primary and secondary equilibria that would be

present if oxine is initially dissolved in an appropriate organic solvent that happens

to be less dense than water. If this solution is added to an aqueous solution that

contains a metal ion such as copper(II) or Cu2+, two immiscible liquid phases persist.

The copper(II) oxinate that initially forms in the aqueous phase, oxine, itself is an

amphiprotic weak acid and quickly partitions into the organic phase. Being amphiprotic means that oxine itself can accept a proton from an acid and can also donate one

to a base. The degree to which oxine either accepts or donates a proton is governed



Ether

phase



Aqueous

phase



HOx



Cu2+ +



CuOx2



2 Ox−



CuOx2



FIGURE 3.6 Distribution of copper oxinate between ether and water.

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by the pH of the aqueous solution. The acidic property is the only one considered

in the development of the equations considered below. The formation of a Cu oxine

chelate can proceed via 1:1 and 1:2 stoichiometry. The fact that it is the 1:2 chelate

that is neutral, and therefore the dominant form that partitions into the nonpolar

solvent, is important. All of the competing primary and secondary equilibria can be

combined to yield a relationship that enables the distribution ratio to be defined in

terms of measurable quantities.

The distribution ratio, D, for the immiscible phases and equilibria shown in Figure

3.6 is first defined as the ratio of chelated copper in the organic phase to the concentration of free and chelated copper in the aqueous phase. Expressed mathematically,

D≡



[CuOx 2 ]o

[Cu ]aq + [CuOx 2 ]aq

2+



Similar to what was done earlier for HOAc, we can define αCU as the fraction

of free Cu2+ in the aqueous phase: then,



α Cu =



[Cu2 + ]aq

2+



[Cu ] + [CuOx 2 ]aq



so that

D=



[CuOx 2 ]o

[Cu 2+ ]/α Cu



(3.17)



Use of αCu is a simple and convenient way to account for all of the many side

reactions involving the metal ion. Substituting the equilibrium expressions into

Equation (3.17) yields



D=



CuOx

2

K D 2 β2 K a [HOx ]2

o

HOx

KD

[H + ]aq



(3.18)



We have assumed that the protonation of HOx as discussed earlier is negligible.

Equation (3.18) states that the distribution ratio for the metal ion chelate LLE

HOx

depends on the pH of the aqueous phase and on the ligand concentration. K D , β2,

and α are dependent on the particular metal ion. This enables the pH of the aqueous

phase to be adjusted such that a selected LLE can occur. One example of this

selectivity is the adjustment of the pH to 5 and extraction as their dithizones to

selectivity separate Cu2+ from Pb2+ and Zn2+.13

The metal chelate LLE was much more common 25 years ago when it was the

principal means to isolate and recover metal ions from aqueous samples of environmental interest. The complexes were quantitated using a visible spectrophotometer

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because most complexes were colored. A large literature exists on this subject.14 The

technological advances in both atomic absorption and inductively coupled plasmaatomic emission spectroscopy have significantly reduced the importance of metal

chelate LLE to TEQA. However, metal chelate LLE becomes important in processes

whereby selected metal ions can be easily removed from the aqueous phase.



11. CAN ORGANIC CATIONS OR ANIONS

BE EXTRACTED?

We have discussed the partitioning of neutral organic molecules from an aqueous

phase to a nonpolar organic solvent phase. We have discussed the partitioning of

metal ions once they have been converted to neutral metal chelates. In this section,

we discuss the partitioning of charged organic cations or charged organic anions.

This type of LLE is termed ion pairing. Ion pair LLE is particularly relevant to

TEQA, as will be shown below. We start by using equilibrium principles and assume

that the only equilibra are the primary ones involving the partitioning of the ion pair

between an aqueous phase and a lighter-than-water organic phase. The secondary

equilibria consist of formation of the ion pair in the aqueous phase. Also, all cations

and anions are assumed not to behave as weak acids or bases. For the formation of

the ion pair in the aqueous phase, we have

C (+aq ) + A (−aq )



K IP

← CA (aq )

→



The ion pair CA, once formed, is then partitioned into an organic solvent that

is immiscible with water according to

CA (aq )



KD

← CA (org )

→



The distribution ratio, D, with respect to the anion for IP-LLE, is defined as

DA − =



[CA ]org

[CA ]aq + [ A − ]aq



In a manner similar to that developed earlier, D can be rewritten as

 K IP [C + ] 





D A− = K D 

+ 





 1 + K IP [C ] 



(3.19)



The distribution ratio is seen to depend on the partition coefficient of the ion

pair, KD, to the extent to which the ion pair is formed, KIP, and on the concentration

of the cation in the aqueous phase. Equation (3.19) shows some similarity to Equation

(3.13).

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12. IS THERE AN IMPORTANT APPLICATION OF IP-LLE

TO TEQA?

Equation (3.19) suggests that if an ion pair that exhibits a high partition coefficient, KD,

forms the ion pair to a great extent (i.e., has a large value for KIP) β, then a large value

for D enables an almost complete transfer of a particular anion to the organic phase.

Of all the possible ion pair complexes that could form from anions that are present in

an environmental sample, the isolation and recovery of anionic surfactants using methylene blue is the most commonly employed IP-LLE technique used in environmental

testing labs today. The molecular structure of this ion pair formed a large organic anion

that is prevalent in wastewater such as an alkyl benzene sulfonate, a common synthetic

detergent, using a large organic cation such as methylene blue, as follows:



O

S

−



O

Tetrapropylenebenzenesulfonate anion

an example of an alkylbenzenesulfonate (ABS)



O−



S



O



O

6-dodecylbenzenesulfonate anion

an example of a linear alkylbenzene sulfonate (LAS)

N



N



S+



Methylene blue cation



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O



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This ion pair absorbs visible light strongly at a wavelength of 652 nm. Because

a method that might be developed around this ion pair and its high percent recovery

into a nonpolar solvent (a commonly used one is chloroform) is nonselective, a

cleanup step is usually introduced in addition to the initial LLE step. Of all possible

anion surfactants, sodium salts of C10 to C20 do not form an ion pair with methylene

blue, whereas anionic surfactants of the sulfonate and sulfate ester types do. Sulfonate type surfactants contain sulfur covalently bonded to carbon, whereas the

sulfate ester type of surfactant contains sulfur covalently bonded to oxygen, which

in turn is covalently bonded to sulfur. A good resource on the analysis of surfactants

in all of its forms, including some good definitions, was published earlier.15 The type

of surfactants that form an ion pair and give rise to a high percent recovery are termed

methylene blue active substance (MBAS). A microscaled version to the conventional

method16 for the determination of MBAS in wastewater is introduced as one of the

student experiments discussed in Chapter 5.



13. ARE THERE OTHER EXAMPLES OF NONSPECIFIC

LLE PERTINENT TO TEQA?

In Chapter 1, the determination of total petroleum hydrocarbons (TPHs) was discussed in relation to EPA method classifications. This method is of widespread

interest in environmental monitoring, particularly as this relates to the evaluation of

groundwater or wastewater contamination. There are several specific determinations

of individual chemical components related to either gasoline, fuel oil, jet fuel, or

lubricant oil that involve an initial LLE followed by a GC determinative step.

Methods for these require LLE, possible cleanup followed by GC separation, and

detection usually via a flame ionization detector (FID). Specific methods are usually

required when the type of petroleum hydrocarbon is of interest. There is almost

equal interest among environmental contractors for a nonspecific, more universal

determination of the petroleum content without regard to chemical specificity. A

sample of groundwater is extracted using a nonpolar solvent. The extracted TPHs are

then concentrated via evaporation either by use of a rotary evaporator, Kuderna–Danish

evaporative concentrator, or via simple distillation to remove the extracting solvent.

The residue that remains is usually a liquid, and the weight of this oily residue is

obtained gravimetrically. An instrumental technique that represents an alternative to

gravimetric analysis involves the use of quantitative infrared (IR) absorption. If the

extracting solvent lacks carbon-to-hydrogen covalent bonds in its structure, then the

carbon-to-hydrogen stretching vibration could be used to quantitate the presence of

TPHs. The most common solvent that emerged was 1,1,2-trichlorotrifluoroethane

(TCTFE). With the eventual total phasing out of Freon-based solvents, the EPA has

reverted back to the gravimetric determinative approach. It is not possible to measure

trace concentrations of TPHs via quantitative IR using a hydrocarbon solvent, due

to the strong absorption caused by the presence of carbon-to-hydrogen covalent

bonds. The author maintains that labs could recycle and reuse the spent TCTFE

without any release of this Freon type solvent to the environment while preserving

the quantitative IR determinative method. Only time and politics will determine

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which method will dominate in the future. Nevertheless, the technique of LLE to

isolate and recover TPHs from water contaminated with oil remains important.



14. CAN LLE BE DOWNSIZED?

We now introduce some recently reported and interesting research that reinforces

the basic concepts of LLE. Jeannot and Cantwell17 have introduced the concept of

a true LLE that has been downsized to a microextraction scale. In the past, the

concept of a micro-LLE (µLLE), as introduced by the EPA and promulgated through

their 500 series of methods, was designed to conduct TEQA on samples from sources

of drinking water. Method 508 required that 35 mL of groundwater or tap water be

placed in a 40-mL vial and extracted with exactly 2 mL of hexane. Organochlorine

pesticides such as aldrin, alachlor, dieldrin, heptachlor, and so forth, are easily

partitioned into the hexane. A 1-µL aliquot is then injected either manually or via

autosampler into a GC-ECD to achieve the goal of TEQA. As long as emulsions

are not produced, this downsized version of LLE works fine. Wastewater samples

are prone to emulsion formation, and this factor limits the scope of samples that can

be extracted by this mini-LLE technique.

Cantwell’s group has taken this scale down by a factor of about 20 to the 1-mL

and below sample volume levels. Some interesting mathematical relationships that

serve to reinforce the principles discussed earlier are introduced here. It does not

matter whether an analyst uses a liter of groundwater sample, a milliliter, or even a

microliter. The principles remain the same.

The principle of mass balance requires that the amount of a solute that is present

in an aqueous sample (e.g., groundwater), amtinitial, remain mathematically equivalent

to the sum of solute in both immiscible phases. Matter cannot escape, theoretically,

that is. If the initial amount of a solute is distributed between two immiscible phases,

an organic phase, o, and an aqueous phase, aq, mass balance considerations require

that

amt initial = amt aq + amt o

We now seek to relate the concentration of solute that remains in the aqueous

phase after µLLE to the original concentration of solute, Cinitial. For example, a

groundwater sample that contains dissolved organochlorine pesticides such as DDT

can be mathematically related to the partitioned concentrations in both phases

according to

C initial =



Vaq C aq + VoC o

Vaq



We would like to express the concentration of analyte in the organic phase, Co,

in terms of the initial concentration of analyte that would be found in groundwater,

Cinitial. Dividing through by Vaq and eliminating Caq for LLE,

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C aq =



Co

KD



gives



C initial



C 

Vaq  o  + VoC o

 KD 

=

Vaq



Substituting for the phase ratio, β, dividing the numerator and denominator by

Vaq, and rearranging gives

 1

β

C initial = C o 

+

KD 1 





Rearranging and solving for Co gives

 KD 

C o = C initial 



 1 + βK D 



(3.20)



Hence, the concentration of solute present in the organic phase can be directly

related to the concentration of solute initially present in the aqueous groundwater

sample, Cinitial, provided the partition coefficient and phase ratio, β, are known. The

reader should see some similarity between Equations (3.20) and (3.16). Equation

(3.20) was derived with the assumption that secondary equilibrium effects were

absent. This assumption is valid only for nonionizable organic solutes.

With these mathematical relationships presented, we can now discuss the experimental details. The end of a Teflon® rod was bored to make a cavity. A volume of

8 µL of a typical organic solvent such as n-octane was introduced into the cavity,

and a cap and rod were fitted to a 1-mL cylindrical vial with a conical bottom, to

which a magnetic stirrer has been placed. After the solvent was placed on top of the

aqueous sample, the sample was stirred for a fixed period at a fixed temperature,

25°C. This enables the solute to diffuse into the organic solvent. A 1-µL aliquot of

this extract is taken and injected into a GC for quantitative analysis. This µLLE

yields a β of 0.008. As values of β get smaller and smaller, the second term in the

denominator of Equation (3.20) tends to zero. For a fixed KD and Cinitial, a low value

for β results in a higher value for Co, and hence a higher sensitivity for this µLLE

technique.

Once a sample preparation method has been established, the analytical methodology, so important to achieving GLP in TEQA, can be sought. The analytical

outcomes discussed in Chapter 2 can now be introduced for this µLLE technique.

An internal standard mode of calibration was used to conduct quantitative analysis using the minivial technique just described. The analyte studied was 4-methyl

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acetophenone and the internal standard was n-dodecane. The slope of the linear

calibration was 4.88 L/mmol, with a y intercept of zero and a coefficient of determination of 0.998.17



15. DOES THE RATE OF MASS TRANSFER BECOME

IMPORTANT IN µLLE?

The kinetics of LLE can also be developed. Kinetics become a more important

consideration when aqueous and organic phases cannot be afforded maximum contact, as is the case when a large sep funnel is used. It is worthwhile to consider

kinetics in the context of the µLLE technique developed by Ma and Cantwell.18 The

general-rate equation for LLE can be written in terms of a differential equation that

relates the rate of change of the concentration of analyte in the organic phase, Co,

to a difference in concentration between the aqueous phase, Caq(t), and the organic

phase Co (t) according to the following:

∂

A

Γ o K D C aq (t ) − C o (t )

Co =

∂t

Vo



(



)



where A is the interfacial area and Γo is the overall mass transfer coefficient with

respect to the organic phase (in units of cm/sec). Thus, the time dependence of solute

concentration in the organic phase can be seen as

C o = C o,equil (1 − e − kt )



(3.21)



Co,equil represents the concentration of solute in the organic phase after equilibrium

has been reached. k is the observed rate constant (in units of sec–1) and is given by

k=



A

Γ o [ K D + 1]

Vo



Combining Equations (3.20) and (3.21) leads to an expression that is significant

to TEQA:

 1 + K Dβ 

C aq,initial = C o (t ) 

− kt 

 K D (1 − e ) 



(3.22)



The term in brackets in Equation (3.22) is usually held constant, and this term

is evaluated by extracting a reference aqueous solution where the concentration is

known. The concentration must, of course, be in the linear region of the distribution

isotherm for both sample and standard.

Cantwell and coworkers have recently extended their µLLE technique to include

a back-extraction using a modification of the minivial discussed earlier. An organic

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liquid membrane that consists of n-octane confined to within a Teflon ring sits on

top of 0.5 or 1 mL of an aqueous sample whose pH is approximately 13 and contains

an ionizable analyte. If an amine is dissolved in water and the pH adjusted to 13,

the amine would remain unprotonated and therefore neutral. A large KD would be

expected, and the amine should partition favorably into the n-octane. A 100- or

200-µL acidic aqueous phase with a pH of approximately 2 is placed on top of the

liquid membrane. The amine is then protonated and back-extracted into the acidic

aqueous phase.19 A further enhancement utilizes a microliter liquid-handling syringe

to suspend a drop of acidic aqueous phase within the n-octane phase. The syringe that

now contains the back-extracted analyte can be directly inserted into the injection

loop of a high-performance liquid chromatograph (HPLC).



16. IS THERE ANY OTHER WAY TO PERFORM LLE?

Yes, indeed. There are several alternatives to separatory funnel LLE, mini-LLE, and

µLLE (just described). Sep funnels are limited to ∼1000 mL or less, while miniLLEs are limited to the size of ~40 mL (such as a typical screw-top cylindrical vial).

For aqueous environmental samples whose volume exceeds 1000 mL, continuous

LLE (C-LLE) is often more appropriate and convenient within which to conduct

LLE. To illustrate, if a 2-L wastewater effluent sample is to be extracted, C-LLE

would be the technique of choice. C-LLE requires a relatively large glass apparatus

whereby the receiving pot can vary in size. C-LLE can be performed using a lighterthan-water extractant or a heavier-than-water extractant. Typical lighter-than-water

extractants include various lower-molecular-weight alkanes such as n-hexane, while

typical heavier-than-water extractants include various chlorinated solvents such as

methylene chloride (dichloromethane).

The operational procedure for lighter-than-water C-LLE has been described from

a manufacturer of C-LLE glassware as follows:20

The aqueous phase to be extracted and a stirring bar are placed in a 24/40 round-bottom

flask. The flask size (up to and including the 5 L) is chosen so that it is not more than

2/3–4/5 full of aqueous phase. The flask is then filled with the lighter-than-water

extracting solvent and gentle stirring is started. The extractor and an efficient condenser

are put into place and a small flask containing an additional portion of the lighter-thanwater extracting solvent is connected to the side-arm and the solvent in the small flask

heated above its boiling point. The solvent vapors distill up the side-arm and condense

at the condenser. The condensed solvent runs down the center tube where it is passed

with stirring, through the aqueous phase. The extracting solvent removes a small amount

of material and separates from the water. Since the density of the extracting solvent is

less than that of water, the solvent rises past the joint at the top of the flask containing

the aqueous phase and, when it reaches the side-arm, it flows back to the distilling

flask though the side-arm. The extracted material then remains in the distilling flask

while the solvent is distilled, condensed and is used o extract again. “Fresh” solvent

is thus used over and over. In this way, by allowing the extractor to operate for long

periods, materials only slightly soluble in the organic solvent can be removed from the

aqueous phase in very high yields and only a relatively small amount of extracting

solvent need be used.



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Heavier-than-water C-LLE designs are operated similarly:20

Some heavier-than-water extracting solvent and a stirring bar are placed in the flask

that contains the aqueous phase to be extracted. A good rule-of-thumb is that the flask

should be about 1/5–1/6 full of heavier-than-water extracting solvent. The extractor is

put in place and, with the aid of a funnel whose stem extends below the side-arm of

the extractor, the aqueous phase to be extracted is added. The aqueous phase will fill

the flask and may move up the vigreux column past the lower return tube. A small

flask containing the heavier-than-water extracting solvent is then connected to the sidearm of the apparatus and the solvent therein heated above its boiling point.



In TEQA, the sample matrix determines whether LLE involving immiscible

solvents is to be used. If the sample is a solid, such as a contaminated soil or

sediment, C-LLE gives way to the Soxhlet extraction apparatus. There have also

been attempts to modify the conventional Soxhlet via miniaturization or instrumentation that pressurizes and heats the extracting solvent. A recent technique promulgated by the EPA is called pressurized fluid extraction. EPA Method 3545 from

Update III of SW-846 has been developed to enable priority pollutant semivolatile

organics to be isolated and recovered from soils, clays, sediments, sludges, and other

solid waste. The Dionex Corporation has developed what they call accelerated

solvent extraction, whereby a much smaller volume of extraction solvent is used. The

vial containing the sample and extracting solvent is both heated and pressurized.

These extreme temperature and pressure conditions supposedly accelerate the rate

at which equilibrium is reached in LLE. The conventional technique for isolating

and recovering semivolatile organics from solid matrices is called Soxhlet extraction.

Soxhlet extraction as an analytical sample preparation technique has been around

for over 100 years. The principle of Soxhlet extraction, abbreviated S-LSE (because

it is a solid–liquid extraction technique), will be discussed in the following section.



17. WHAT IS SOXHLET EXTRACTION ANYWAY?

A solid matrix of environmental interest, such as soil that is suspected of containing

any of the more than 100 priority pollutant semivolatiles, is placed into a cellulosic

thimble. Vapors from heating a volatile organic solvent rise and condense above the

thimble. This creates a steady-state condition called reflux. The refluxed solvent

condenses into the thimble and fills until it overflows back into the distilling pot.

Reflux is a common technique in organic chemistry and serves to bring the S-LSE

process to a fixed temperature. Thus, solutes of interest are able to partition between

a fixed weight of contaminated soil and the total extractant volume. Usually, a series

of six vessels with six separate heaters are available as a single unit whereby the

incoming and outgoing water lines for the reflux condensers are connected in series.

A large phase ratio is obtained. S-LSE is usually conducted for one sample over a

period of 12 to 24 h. An overnight continuous S-LSE is quite common. After the

extraction time has ended, the glass S-LSE vessel is cooled to room temperature.

The extractant in the thimble is combined with the refluxed extractant in the distilling



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