sampleprepcompend kak050-kemm06 ht 2014-v21-slutlig
TRANSCRIPT
Sample preparation, KAK050/KEMM06, HT 2014
SamplePrepCompend KAK050-KEMM06 HT 2014-v21-SLUTLIG.docx Senast sparat 2014-‐11-‐03 20.43
SAMPLE PREPARATION IN CHROMATOGRAPHIC ANALYSIS Extraction Karl-‐Gustav Wahlund Centre for Analysis and Synthesis Department of Chemistry Lund University
Contents Introduction ............................................................................................................... 2
Basics of sample preparation ..................................................................................... 3
Fundamentals of extraction ....................................................................................... 6 Solvent extraction .............................................................................................................. 6
Liquid-‐liquid extraction (LLE) ................................................................................................. 7 Fraction extracted ................................................................................................................. 8
Batch extraction ................................................................................................................ 12 Single batch extraction ........................................................................................................ 12 Repeated batch extraction .................................................................................................. 12
Continuous extraction ....................................................................................................... 13 Extraction techniques and applications ............................................................................. 14
Techniques .......................................................................................................................... 14 Applications ......................................................................................................................... 15
Octanol/water distribution constants, KD,o/w ................................................................................. 15 Solid phase extraction (SPE) ........................................................................................................... 16
Quantitative treatment of liquid-‐liquid extraction .................................................... 21 A neutral nonprotic analyte .............................................................................................. 21
Distribution constant ........................................................................................................... 21 What determines the value of the distribution constant, KD? ....................................................... 22
Distribution ratio ................................................................................................................. 24 More about total concentrations ........................................................................................ 25
A neutral weak organic acid HX ......................................................................................... 26 A neutral weak organic base B .......................................................................................... 29 How to use the log D – pH diagrams (log-‐log plots) ............................................................ 31
Applications of liquid-‐liquid extraction ..................................................................... 32 Separation of acids and bases by single batch liquid-‐liquid extraction ............................... 32
Two neutral weak acids, HX and HY .................................................................................... 32 Two neutral weak bases, codein and papaverin ................................................................. 33
LITERATURE ................................................................................................................ 33
2
Introduction
Sample preparation is a most important and indispensable part in any quantitative analytical determination method. It has a decisive influence, in fact cardinal, on the quality of the reported result, consequently the accuracy and precision. Without knowing these it can be risky, even meaningless, to try to draw any advanced conclusions on the interpretation of the result, for example as a basis for decisions on various issues. Sample preparation includes procedures known as pre-‐treatments of the original sample. They are necessary before the final quantification of the intended sample analyte can be made. The samples can be of a large variety such as body fluids and tissues, waste-‐water, environmental polluted aquatic waters, environmentally spread chemical compounds, chemically engineered nanoparticles, etc. Each sample type requires its specific pre-‐treatment.
The general aspects of sample preparation are often described in textbooks for undergraduate students, such as your own, Quantitative Chemical Analysis by D.C. Harris. In chromatographic analysis the aim of sample preparation is to obtain the analyte in a homogeneous solution that can be injected onto the chromatographic column. The solvent for the analyte needs to be compatible with the mobile phase and the analyte solution obtained needs to be free from interfering compounds. For example, in GC the analyte solvent has to be vaporizable when introduced into the sample injector. In column LC the solvent needs to be completely miscible with the mobile phase solvent.
One of the most used sample pre-‐treatments in chromatographic analytical methods, as well as many, many other, is the extraction of the analyte from the sample into a suitable solvent. For this reason, and also due to the very widespread use of extraction in general, the present text is aimed to assist in your understanding of the theory, principles and applications of extraction. Once you understand these you will be well prepared to deal with the multitude of different extraction techniques that exist, and most importantly, continue to be developed. You will find that the fundamentals of extraction are applicable to nearly all those techniques. They are not covered in any detail here since they are described in your course-‐book, which is recommended for study. In addition, course lectures, lecture notes, and exercises add to the understanding.
Care has been taken to define and explain concepts, terms and quantities and apply modern terminology and symbols following the recommendations by the International Union of Pure and Applied Chemistry (IUPAC). However, exceptions occur where confusion needs to be avoided. In some cases these exceptions lead to deviations from many textbooks since it can take a long time before newer terms and symbols find their way into the literature. Clearly, the terminology in chemistry has a very long history and tradition, at least from the 1800s, and what you find today as recommendations by IUPAC is a reflection of this long tradition. Unfortunately, the last IUPAC recommendation 1993 on liquid-‐liquid distribution (solvent extraction) has caused confusion. In the present text an attempt is made to correct this and straighten it out.
To assist in your understanding and interpretation there are inserted in the text many citations
3
from literature sources such as the IUPAC recommendations and on-‐line dictionaries (Wikipedia, www.thefreedictionary.com, and more). Reading the original literature source, rather than re-‐interpretations and re-‐phrasings in textbooks, course-‐books, and course compendia, helps your understanding of the concepts, terms, quantities and symbols. The citations are inserted immediately after that a certain term occurs in the body text. You can choose to read them immediately, or return to them later.
Useful on-‐line sources for terms and words are: Swe: Ordböcker • Svenska Akademiens ordlista • Svenska Akademiens ordbok http://www.sprakradet.se/2130
Dictionaries Oxford English Dictionary Encyclopedia Encyclopedia Britannica Encyclopedia Britannica (http://www.britannica.com/)
Since many technical and chemical terms originate in Latin or Greek an even better way of understanding is to look for the original Latin or Greek semantic meaning. This same meaning is very often the essence of the terms as we use them today.
Basics of sample preparation As mentioned the sample may be subjected to a series of pre-‐treatments. These are integral parts of the sample preparation and are performed in a certain sequence. This pre-‐treatment sequence often comprises four consecutive aims and they can be achieved by four corresponding techniques:
Sample pre-‐treatment sequence
Aim Technique/Common term 1. Isolation Extraction
2. Purification Clean-‐up
3. Separation Chromatography
4. Detection Detector
The first pre-‐treatment serves to isolate the analyte from the main material of the sample, i.e. the matrix.
What is isolate/isolation? Isolate (verb): place or set apart, obtain in pure form, set apart from others. Context example: The chemist managed to isolate the compound.
4
Source: http://www.audioenglish.net/dictionary/isolate.htm. Obtain or extract (a compound, microorganism, etc.) in a pure form. Source: http://www.oxforddictionaries.com/definition/english/isolate
Isolation [verb]: the act of isolating something; setting something apart from others. Source: 1 Click Oxford Dictionary. Isolate (noun): A person or thing which has been or become isolated. Source: http://www.oxforddictionaries.com/definition/english/isolate.
What is a matrix? Matrix n., pl. matrices or matrixes; swe. matris: a substance, situation, or environment in which something has its origin, takes form, or is enclosed. Source: http://www.thefreedictionary.com
If the matrix is complex, i.e. contains many different compounds at different levels of concentrations or amounts, the isolate may hardly become pure in the analyte. For complex matrices the aim of the isolation at this stage is to remove the analyte from the majority of the matrix compounds. This isolation can commonly be made by extraction of the analyte from the matrix.
What is extraction? Extraction means that an object is "drawn out" from its environment (lat. extrahere "to draw out (of/from)" from 'ex' for out and 'trahere' for draw; swe. "dra ut"). Source: http://www.thefreedictionary.com
What is to extract? To transfer a solute from a liquid phase to another immiscible or partially miscible liquid phase in contact with it. Note: The term is also applied to the dissolution of material from a solid phase with a liquid in which it is not wholly soluble (i.e. leaching). Source: PAC, 1993, 65, 2373, Nomenclature for liquid-‐liquid distribution (solvent extraction) 1.8, IUPAC Recommendations 1993.
If the isolate, the extract, will contain other components than the analyte so that they will interfere in the analysis, the extract needs to be subjected to some purification to remove the interferents. The term clean-‐up (swe. upprening) represents various methods by which the purification can be obtained. Interferences can occur in the subsequent chromatographic separation-‐detection.
What is an interferent (noun)? (Noun) interferent (plural interferents) (chemistry), swe. störande ämne. Any substance whose presence interferes with an analytical procedure and generates incorrect results. Source: http://en.wiktionary.org/wiki/interferent
What is an interference (noun)? Swe. störning.
In chromatographic separations column interferences can be caused by
1. compounds that cannot be separated from the analyte in the column, 2. other sample components, even the solvent itself, that causes some damage
to the column, such as precipitation of solids.
5
Case 1. is counter-‐acted by an increase the separation selectivity (α) of the column, supposing that the separation efficiency (N-‐value) already has been maximised. Please note that this case presupposes that the interferences are also detection interferences. Case 2. is counter-‐acted by a suitable of choice of the nature and type of the mobile phase solvent in relation to that in the injected analyte solution.
When no remaining interfering compounds exist the final chromatographic separation and detection can be achieved, resulting in the quantification (quantitative determination).
Additionally, the extraction can be effectively used to obtain a pre-‐concentration of the analyte, which means that its concentration is increased relative to in the original sample. This occurs if the analyte is extracted into a much smaller volume than that of the original sample. Pre-‐concentration has one very important use:
Sometimes two or more of the methods are united in one, for example if the extraction already suffices the need for purification.
The composition of the matrix has a strong influence on the need for isolation and purification. Matrices can be from quite simple to very complex. A simple water solution, or an organic solvent solution, of the analyte-‐containing sample represents perhaps the simplest matrix but it can yet contain a lot of compounds that may be closely related to the analyte and interfere in the separation -‐ detection. More complex matrices are represented by various environmental, food, biological, biochemical, biomedical, and pharmaceutical/medical samples. Blood, blood plasma, and urine samples are in focus in drug discovery and development in the pharmaceutical industry. In the food industry raw materials from animal and plants may occur. Such complex sample matrices can be liquids (solutions), liquid suspensions (dispersions), or solids. A liquid sample can contain dissolved components as well as suspended solid components.
When the matrix is a solid, or contains insoluble matter, the isolation and purification of analytes can be very challenging. This is common for plant, food and soil samples, especially if the analyte is strongly bound to, or captured, in structures within the solid matrix, or if it is insoluble itself in common extraction solvents. High molar mass compounds such as starch, dietary fibres, and proteins require specially designed methods for their isolation. However, most of the remainder of this text will deal with common low molar mass substances.
To enrich (swe. anrika) the analyte so that its concentration increases and the detector signal becomes higher for a given sample volume injected. This is especially important in trace analysis (swe. spåranalys).
6
Fundamentals of extraction Extraction is in itself a separation in which the analyte becomes separated from its matrix.
What is separation? “Separation process: In chemistry and chemical engineering, a separation process, or simply a separation, is any mass transfer process used to convert a mixture of substances into two or more distinct product mixtures, at least one of which is enriched in one or more of the mixture's constituents. In some cases, a separation may fully divide the mixture into its pure constituents. Separations are carried out based on differences in chemical properties such as size, shape, mass, or chemical affinity between the constituents of a mixture, and are often classified according to the particular differences they use to achieve separation. In the case that no single difference can be used to accomplish a desired separation, multiple processes will often be performed in combination to achieve the desired end". Source: Wikipedia, the free encyclopedia.
Solvent extraction
(Swe. lösningsmedelsextraktion) (Alternative term: liquid extraction; swe. vätskeextraktion)
This procedure is technically simple. Just add an immiscible solvent to a liquid sample (solution or dispersion), or a solid sample, and shake the mixture, which means to homogenise it. Any sample component that has enough solubility in the solvent can then be extracted. For small organic analytes, an organic solvent would be preferred. For solid samples, either an aqueous or an organic solvent can be used, depending on whether the sample analyte is water-‐soluble or not. Common solvents for extraction are water, ethanol, ethyl acetate, diethyl ether, dichloromethane, toluene, and hexane.
What is solvent extraction? The process of transferring a substance from any matrix to an appropriate liquid phase. If the substance is initially present as a solute in an immiscible liquid phase the process is synonymous with liquid-‐ liquid extraction. Source: IUPAC Compendium of Chemical Terminology 1399 of 16221) and PAC, 1993, 65, 2373, Nomenclature for liquid-‐liquid distribution (solvent extraction), IUPAC Recommendations 1993, 1.19, page 2379. Note: 1. If the extractable material is present in a solid (such as a crushed mineral or an ore) the term leaching may be more appropriate. The extractable material may also be a liquid entrapped within or adsorbed on a solid phase. 2. Common usage has established this term as a synonym for liquid-‐liquid distribution. This is acceptable provided that no danger of confusion with extraction from solid phases exists in a given context. Source: PAC, 1993, 65, 2373 (Nomenclature for liquid-‐liquid distribution (solvent extraction) (IUPAC Recommendations 1993)) on page 2379.
Solvent extraction has a long history in preparative organic chemistry to isolate various compounds from natural matrices. For example, ethanol extraction can be used to obtain extracts of various spices and other compounds in plants. In the pharmaceutical/pharmacological area, amines, for example alkaloids, can be extracted from plants using an organic solvent, one example being the extraction of morphine from the dried milky exudate of the unripe seed capsule of the opium poppy. However, efficient extraction of
7
an alkaloid requires basic conditions so that the alkaloid is present in the neutral basic form that is soluble in the organic extraction solvent. Basic conditions are obtained by adding an aqueous alkali solution to the sample matrix and then performing the extraction.
In preparative inorganic chemistry solvent extraction has been used for long time to extract metals from minerals by leaching (swe. lakning) and metal ions from soil in environmental and ecological investigations, sometimes as complexes with inorganic acids or with chelating agents.
Liquid-‐liquid extraction (LLE)
Solvent extraction of an aqueous sample by an immiscible organic solvent, or solvent mixture, technically refers to an extraction of the sample analyte from an aqueous phase to an organic phase. This is known as liquid-‐liquid extraction (LLE). Common alternative terms are liquid-‐liquid distribution and liquid-‐liquid partition.
What is distribution? n. the act of distributing, apportionment, division into categories, a thing or portion being distributed, arrangement or location; swe. fördela. Source: http://www.thefreedictionary.com; The apportionment of a solute between two phases. Note: The term partition (1.15) or extraction (1.9) may also be used in this sense where appropriate. Source: IUPAC Recommendations 1993 on Nomenclature for liquid-‐liquid distribution (solvent extraction) (1.4); PAC, 1993, 65, 2373.
What is partition and partitioning? n. v. The state of being so divided. To divide into parts, pieces, or sections. The act or process of dividing something into parts; swe. dela in, indela, dela upp, uppdela. Source: http://www.thefreedictionary.com.
What is liquid-‐liquid distribution (extraction) (partition)? The process of transferring a dissolved substance from one liquid phase to another (immiscible or partially miscible) liquid phase in contact with it. Note: Although extraction, partition and distribution are not synonymous, extraction may replace distribution where appropriate. Source: IUPAC Recommendations 1993 on Nomenclature for liquid-‐liquid distribution.
What is an extract (noun)? The separated phase (often but not necessarily organic) that contains the material extracted from the other phase. Notes: Where appropriate the term "loaded solvent" (4.15) may be used, but is not recommended. Source: IUPAC Recommendations 1993 on Nomenclature for liquid-‐liquid distribution (solvent extraction) (2.7).
What is an extractant? The active component(s) primarily responsible for transfer of a solute from one phase to the other. Notes: (i) The term extracting agent is a synonym but solvent (2.12) and ligand should not be used in this context. Source: IUPAC Recommendations 1993 on Nomenclature for liquid-‐liquid distribution (solvent extraction) (2.8).
What is a modifier?
8
A substance added to a solvent (2.12) to improve its properties e.g. by increasing the solubility of an extractant (2.8), changing interfacial parameters, or reducing adsorption losses. Source: IUPAC.
What is the solvent (in liquid-‐liquid distribution)? The term applied to the whole initial liquid phase containing the extractant (2.8). (2.12?). Source: IUPAC.
For quantitative chemical analysis (quantitative determination) of the analyte in the original sample (the feed phase), it is necessary to obtain a complete transfer of the analyte from the feed into the extract (complete extraction = quantitative extraction) and preferably minimize the extraction of other sample components that might interfere in the following chromatographic separation or the detection. This means that the extraction conditions may need to be optimised. For this purpose it is important to have tools for systematic regulation of the fraction extracted of the analyte in the sample. This requires knowledge of the parameters that can influence the liquid-‐liquid distribution equilibrium of the analyte molecules between the two phases. The possibilities are enormous since the composition of both liquid phases can be changed in many ways. Among these is, for example, a change of the pH in the aqueous phase (if analytes are acids (protogenic) or bases (protophilic), in other words, protic molecules) and using mixtures of different organic solvents, or adding complex forming agents to the organic phase.
Fraction extracted
The fraction extracted, fE, expresses how large a fraction of the sample analyte, originally present in the aqueous phase, that is extracted into the organic phase. The fraction extracted is defined by
!! = !!!!!
where QA is the amount (quantity) of analyte A found in the organic extract and QoA is the
amount originally present in the aqueous phase, hence the sum of the amount of A in the two phases after equilibration. For example, if nearly all amount is extracted the fraction extracted will be nearly 1. This corresponds to a percentage fraction extracted of nearly 100%.
What is equilibration? The operation by which a system of two or more phases is brought to a condition where further changes with time do not occur. From: PAC, 1993, 65, 2373, Nomenclature for liquid-‐liquid distribution (solvent extraction), IUPAC Recommendations 1993 1.6
What is the fraction extracted? The fraction of the total quantity of a substance extracted (usually by the solvent) under specified conditions, i.e. where QA is the mass of A extracted and Qo
A is the total mass of A present at the start. Notes: 1. fE may be expressed as a percentage, %fE. 2. The term extractability is qualitative and should not be used as a synonym for fraction extracted. 4. The fraction extracted is also known as the recovery factor, especially for a multistage process.
9
Source: Nomenclature for liquid-‐liquid distribution (solvent extraction) PAC, 1993, 65, 2373 and IUPAC Recommendations 1993, page 2384.
The percentage fraction extracted, %f!, of the analyte after one single extraction can be calculated by
%!! =100
1+ !!"!! !!"#
=100
1+ 1!! !
where !!" and !!"# are the volumes of the aqueous and organic phases and r is the phase volume ratio defined by ! = !!"# !!". DA is the so-‐called distribution ratio for A as defined by
Capital C denotes the total concentrations (of A) in each of the two phases. The distribution ratio and the concept of total concentration will be discussed in detail in further sections below. Please note already here that the equation for the percentage fraction extracted is asymptotic and actually has two asymptotes. The relevance of this will be detailed below.
What is the distribution ratio (in liquid-‐liquid distribution)? The ratio of the total analytical concentration of a solute in the extract (regardless of its chemical form) to its total analytical concentration in the other phase. Source: IUPAC (2.7, 3.5)
What is the phase ratio (r) (in liquid-‐liquid distribution)? The ratio of the quantity of the solvent to that of the other phase. Note: Unless otherwise specified the phase ratio refers to the phase volume ratio. Source: IUPAC (2.12; 3.19).
In summary the fraction extracted depends on the
These two are the parameters that can be used to regulate the % fraction extracted to the desired level. The most powerful parameter is the distribution ratio and to have some idea of its value makes possible a much more intelligent use of the method of extraction than otherwise. It is therefore essential to know what governs its value and from there conclude how it can be regulated. Clearly, the most accurate and successful planning of an extraction requires that DA is known. The phase volume ratio has a somewhat more limited usefulness
DA =CA,org
CA,aq
1) analyte distribution ratio
2) phase volume ratio.
10
since it will be rather impractical to use phase volume ratios >100 and <0.01. However, inside these limits it is an equally important regulation parameter as the distribution ratio.
Technically, this way of performing the liquid-‐liquid extraction technique is called batch extraction (swe. satsvis extraktion) and tools to use are small extraction tubes, preferably centrifuge tubes, large separatory funnels of a size depending on the volumes, or Erlenmeyer flasks (shake flasks).
In quantitative analytical determinations it is essential to know how to obtain complete extraction, also called quantitative extraction (swe. fullständig extraktion, kvantitativ extraktion) and negligible extraction (swe. försumbar extraktion) for a given analyte. Complete extraction means that nearly all amount of the analyte will be extracted. Negligible extraction means that nearly zero amount is extracted, which means that nearly everything remains in the aqueous phase (this condition can be used for so called back-‐extraction of the analyte to an aqueous phase after an initial quantitative extraction to the organic phase). Ideally, all other sample components than the analyte should be subject to negligible extraction. This means that the only compound extracted would be the analyte. Such a situation represents the ultimate level of extraction selectivity, however, which is very rare in practice for complex samples. The consequence is often that the extract has to be subjected to chromatographic separation with selective detection.
The way that the % fraction extracted depends on the distribution ratio !! is fundamental and experimentally highly important. The dependence can be calculated from the equation above for %f!, which is graphically illustrated in the following Diagram for the case of “equal phase volumes”, Vorg = Vaq ; r = 1.
Diagram The dependence of the percentage fraction extracted on the distribution ratio at equal phase volumes.
0
20
40
60
80
100
0,001 0,01 0,1 1 10 100 1000
Distribution ratio (DA)
% F
ract
ion
extr
acte
d
11
The consequences of the asymptotic nature of the equation are clear in the diagram. It means that complete extraction defined as exactly 100% fraction extracted is impossible (there will always be some molecules left in the aqueous phase). Likewise, negligible extraction in terms of exactly 0% fraction extracted is also impossible.
The highly important Diagram shows that at DA = 1, where the % fraction extracted is 50 %, the steepness of the curve is high. This means the % fraction extracted is very sensitive to the value of the distribution ratio. Therefore, even minor variations in DA can lead to large deviations in the % fraction extracted. This potentially results in a non-‐robust extraction method. Instead, robust conditions should be sought for. Clearly, they exist at high distribution ratios where the gradient of the function starts to decrease, for example at !! ≥ 10 where the % fraction extracted is ≥ 90%.
Of course, if a complete extraction is wanted, such as for a quantitative determination of an analyte originally present in the aqueous phase, or, in a preparative extraction when a high yield is wanted, the distribution ratio should be chosen to give, for example (please note: it is Your choice!)
%f! ≥ 90%, if a 10% loss can be accepted,
%f! ≥ 95%, if a 5% loss can be accepted, and
%f! ≥ 99%, if a 1% loss can be accepted.
In quantitative determinations these values represent accuracies, in terms of percentage relative errors, of -‐10%, -‐5%, and -‐1%, respectively.
It is up to you as the analyst to decide what level of accuracy that is required for a certain quantitative determination.
Therefore, the definition of “complete” and “negligible” will need to be arbitrary depending on the requirements in the actual case and for what purpose the results are going to be used. Generally, however, there is hardly any reason to strive for more than 99% fraction extracted since already the random experimental relative errors are higher than this. Instead, 95% should suffice in most cases. Again: it is your choice!
Anyway, in general discussions it may be suitable to define
In cases where only rather low distribution ratios can be obtained (primarily due to limitations in increasing !! enough much) the possibility to use high phase volume ratios Vorg/Vaq can be
complete extraction as when %!! ≥ 99%
negligible extraction as when %!! ≤ 1%
12
used. For example, if only DA = 10 can be obtained for a certain compound, then only 91% will be extracted using equal phase volumes. But if the phase volume ratio Vorg/Vaq is increased to 10, then 99% will be extracted. However, using very high phase ratios will be experimentally rather impractical. The upper limit may be at about Vorg/Vaq = 100.
When equal phase volumes, Vorg = Vaq, are used the requirements on the distribution ratio to give complete and negligible extraction, respectively, are:
These numbers may be useful to remember.
Batch extraction
(Swe. satsvis extraction) (Alternative terms: static extraction, equilibrium extraction)
Batch extraction means that one portion (volume) of the aqueous phase is homogenised (mixed) with one portion of the organic phase. This makes one batch. The extraction will eventually come to equilibrium after enough time and intensity of homogenisation. Then the extraction is finished. The equilibration time depends on the efficiency of the convection during the homogenisation and on the rate of the extraction equilibrium (kinetics).
The alternative terms static extraction and equilibrium extraction mean that the extraction has come to equilibrium so that there will be no further changes of the analyte concentration in the aqueous and organic phases. The extraction has then reached static conditions.
Single batch extraction
This was treated above under Fraction extracted.
Repeated batch extraction
Exercise 1 1. What level of distribution ratio must be exceeded if quantitative extraction would be obtained using equal phase volumes? 2. What is the maximum distribution ratio allowed when an analyte shall be negligibly extracted using equal phase volumes. 3. In a single extraction experiments using equal phase volumes it was noted that the %extraction yield (%fraction extracted) was only 25%. What conditions can be used to obtain 99% without changing the composition of the aqueous and organic phases.
complete extraction (%fE ≥ 99 %) requires !! ≥ 100
negligible extraction (%fE ≥ 1 %) requires !! ≤ 0,01
13
(Alternative terms: repeated static extraction, repeated equilibrium extraction)
What is the fraction extracted? Continued from above. Notes: 3. If the aqueous phase is extracted with successive portions of solvent, the phase volume ratio (solvent/feed) being r each time, the fraction extracted is given by: /See equation below in this compendium/. Source: Nomenclature for liquid-‐liquid distribution (solvent extraction), IUPAC Recommendations 1993, page 2384.
When the distribution ratio is so low that a complete extraction cannot be obtained in a single extraction it is possible to increase the fraction extracted by repeated extractions. This means that the remaining fraction in the aqueous phase will be subject to another extraction by adding fresh solvent of the same volume as in the first extraction. Before this, the first extract has to be separated from the aqueous phase and collected. The following equation can be used to calculate the percentage fraction remaining of analyte in the aqueous phase after n extractions:
%!!"#,! = 100
1 + !! !!"#!!"
!
When the percentage remaining reaches 1% a complete extraction has been obtained. The separated organic phases will then be united to collect the 99% extracted. For practical reasons the upper limit of the number of extractions may be at n = 3 – 4.
Alternatively, a direct calculation of the percentage fraction extracted can be made by the following equation:
%!! = 100 1− 1 + !!!!"#!!" !!
Continuous extraction
(Alternative terms: dynamic extraction, non-‐equilibrium extraction)
Since repeated batch extraction can become unpractical when the distribution ratio is very small (requiring a high number of batch extractions) a more efficient way is to use continuous extraction. It can be regarded as integrated repeated batch extraction using a high value of n. Continuous extraction fits best for solid samples, hence in solid-‐liquid extraction (meaning that
Exercise 2 What will the percentage extracted to the organic phase, %fE,n,org , be if 1) the distribution ratio was DA = 1, the phase volumes were equal, and the number of extractions were n=1, n=2, and n=4, respectively? 2) the distribution ratio was DA = 3, everything else the same.
14
a solid is extracted by a liquid). One way to perform it is that fresh extraction solvent be supplied continuously to the sample. Then the technique is known as continuous flow extraction. There is a plentiful of techniques that have been designed to perform continuous extraction. Perhaps the simplest is column extraction. The ground solid sample is filled in a glass tube and the extraction solvent is let flow through the bed by gravity. A similar everyday example is the extraction of caffeine when preparing brewed coffee on a filter paper. In this case the extraction solvent is boiling water. Another possibility occurs if the solvent can be evaporated. Then, after one extraction, it can be distilled, condensed and passed again to the extraction flask, and so on over and over again. Many different apparatuses have been designed for this purpose, for example the Soxleth apparatus.
For complex solid samples it is very difficult, nearly impossible, to calculate the number of batch extractions needed for complete extraction. The reason is that solid phases do not follow, other than perhaps qualitatively, a behaviour corresponding to that of aqueous solutions in liquid-‐liquid extraction. It is usually impossible to determine, in advance and separately, a quantity that corresponds to the distribution ratio as applied in LLE. Therefore it is impossible to predict the fraction extracted. In such cases, repeated batch extraction is at risk of giving only partial extraction. The chances are better by continuous extraction.
However, it is impossible to predict how far the continuous extraction needs to be driven in order to reach complete extraction1. Hence, empirical knowledge is needed to guide in the work. If well performed complete extraction may yet be obtained. Then the extraction needs to go on until nearly no more analyte remains in the sample. It is obvious that extraction can only be obtained for the part of the analyte that really is extractable. This may not be so in solid samples when some unknown fraction can be buried in the deeper structure of the solid material so as to be practically non-‐extractable due to too strong binding to, or too low mass transport rate within the solid. Sometimes therefore a distinction is made between the extractable and the non-‐extractable fraction of the total amount of analyte in the solid sample. Since the non-‐extractable fraction is usually unknown any assessment of the completeness of the extraction only refers to the extractable fraction.
Extraction techniques and applications
Techniques
1 Sometimes the term ”exhaustive extraction” is used (swe. uttömmande). It means that the fraction extracted has been increased so much, either by repeated batch extraction or continuous extraction, so that complete extraction should have been reached.
Liquid-‐liquid extraction (LLE) Solid phase extraction (SPE) Liquid-‐solid extraction (LSE) Solid-‐liquid extraction (SLE) Supercritical fluid extraction (SFE)
Solid-‐phase microextraction (SPME) Liquid-‐phase microextraction (LPME) Supported liquid membrane extraction (SLME) Hollow fibre liquid phase
Pressurised liquid extraction (PLE) Pressurized fluid extraction (PFE) Membrane extraction (ME) Electromembrane extraction (EME)
15
To perform extractions many different extraction techniques are available. The list below
covers many of them, the most established as well as newer ones. Many of these are described in regular textbooks. It should be noted that in some cases the sample may be a liquid but the extractant a solid, such as in liquid-‐solid extraction and porous membrane extraction. No matter, the basic concepts of solvent extraction described above are applicable, for example regarding the fraction extracted.
Applications
Octanol/water distribution constants, KD,o/w For several decades researchers have collected experimental data for the distribution constants of a large number of organic compounds in the phase system 1-‐octanol/water. The idea has been that the lipophilic 1-‐octanol solvent would have a similarity to the biological membranes and tissues that foreign compounds have to pass in order to enter into an animal or human body. If therefore a foreign compound has a high KD in 1-‐octanol/water, symbolized by KD,oct, or KD,o/w, it is thought that the compound therefore can enter into an animal or human body. The data are usually presented in logarithmic form, for example as log KD,oct or log KD,o/w.
In the literature the logarithmic octanol/water distribution constants are commonly presented as “log P” values. This goes back to a publication by Leo, Hansch, and Elkins in 1971. The P comes from “partition coefficient” which is an alternate term for distribution constant and which has been frequently used in the past, and still is. However, KD represents a more strict terminology and will be used in this text.
One area where log KD,o/w data have been used is in environmental science where it is necessary to try to predict how well lipophilic compounds can penetrate into the environment, especially various potentially toxic lipophilic compounds.
Another area is in pharmacology and medicinal chemistry where log KD,o/w data are used to estimate the lipophilicity of compounds intended for use as drugs. A high value is thought to be proportional to the tendency to reach the intended target in the body, how strong the effect of the drug will be, and how long it will remain in the body in the active form. However, many biologically active compounds are ionized (due to acid-‐base equilibrium in water) in the body fluids and then the log Do/w data may be more relevant. For this it is necessary of course to know the pKa value of the compound and the pH in the body fluid. Determination of log KD,o/w is one of the standard measurements that authorities require for any new drug substance. Obviously, the animal body is so complex that only a log KD,o/w is not enough to describe its biological availability. For this reason distribution data for transport across certain standard biological membranes are also demanded.
microextraction (HFLPME)
16
Much work has been done to correlate the log KD,o/w data to the physiological effect of the substance. In fact, this has been the driving force behind the development of the octanol/water distribution constant data. The correlation work goes under the term Quantitative Structure-‐Activity Relationship (QSAR), or more generally Quantitative Structure-‐Property Relationship (QSPR). Therefore there was a great need for log KD,o/w data and to satisfy this need a lot of interest has been put into prediction of log KD,o/w data so that the experimental work can be limited or that data can be predicted for substances that perhaps have not yet been synthesized. This knowledge has been put into computer software which can predict the log KD,o/w for any wanted structure.
Solid phase extraction (SPE) (Swe. fastfasextraktion)
Solid phase extraction is one of the most common techniques to purify, isolate and pre-‐concentrate substances that are present in aqueous samples before they are subjected to chromatographic separation. Very short extraction columns packed with relatively large-‐diameter (> 37 μm) particles (solid phase) that give a low flow resistance are utilized. This means that a liquid flow can be driven by gravity, or by a small pressure differential obtained by suction using a vacuum line. It is the solid phase that is the extractant in this case, hence a liquid-‐solid extraction. The process is of fundamental interest since it has other applications. The main result is a concentrating effect, of analytes at the top of analytical columns and the use of this phenomenon for application of large samples volumes on the columns. In this context, large sample volumes refer to volumes much larger than the column itself. Before going into the details it is necessary to derive some important governing relationships.
Derivation of the starting zone volume of the analyte The following Figure illustration explains in chromatographic terms what is going on during the introduction of a sample of volume !! into an LC column if the sample solvent is of lower elution strength than the regular eluent (the column mobile phase). During the sample loading, once inside the column, the sample solvent itself acts as the mobile phase causing the analyte to obtain a certain retention factor !!. The sample solvent is chosen so that it gives a much higher retention factor than the regular eluent.
1 2 3
Eluent
17
Figure. Illustration of the concentrating effect. In 1. a sample of volume !0, containing an analyte dissolved in the sample solvent, is to be introduced into the column. The sample volume corresponds to a zone width of !0. 2. After, in this case, ~ 25% of the sample volume remains to be introduced into the column the analyte has started to become concentrated (enriched) at the top of the column since it migrates under conditions of high retention, hence at low transport velocity, !!. This is much lower than the eluent (mobile phase) velocity itself, !. Within the column, therefore, the analyte occupies only a short distance, the enriched zone. In 3. the !!"#$" is the volume of sample solvent that the analyte is dissolved in after complete enrichment, right before the start of the elution. Note the slightly increased width of the enriched zone.
For the situation illustrated in the Figure, the expressions below can be obtained.
Definitions !! = retention factor of the analyte during sample application, hence when the acting mobile phase is the sample solvent itself,
!! = sample solution volume,
!! = sample solution zone width in length units,
!!"#$" = analyte zone width in volume units when the sample has been completely introduced into the column, i.e. when the complete sample volume, but not more, has been loaded. This represents the starting conditions before the following elution by the eluent, hence the analyte starting zone,
!! = length of the sample solvent zone in the column when the complete sample volume has been applied; this is the distance into the column that the sample solvent has been introduced in order to apply the full amount of analyte on the column,
!!! = length of the analyte starting zone, i.e. the distance over which the analyte is distributed when sample application is finished,
! = sample application time (the time it takes to introduce the sample plug completely, but not more, into the top of the column).
Derivation The above means that when a sample plug is being introduced into the column the analyte retention is governed by the composition of the solvent making up the sample solution. In other words, the sample solvent itself is the acting mobile phase. If this solvent causes a very high retention factor, the analyte will migrate slowly into the column, much slower than the sample solvent itself is transported. Therefore the analyte will be transported into the column a much shorter distance than the solvent. The analyte migration velocity and the distance migrated is ruled by the standard chromatographic relationships. The result is that the analyte becomes distributed over a much shorter distance than the sample solvent. Comparing with the original sample volume the analyte has become concentrated into a much smaller volume.
18
This represents an in increase in its concentration. This is the concentrating effect, hence enrichment.
Please consider that
!!! ∝ !!"#$"
!! ∝ !!
!! = !!!
! analyte linear velocity (distance/time)
! = !!
! eluent/mobile phase linear velocity
!!! =
!!!
!′
but also that
!!!= !
!!!! ⇒ !
!!
!!= !
!!!!
and therefore
and
The final two equations show, in a nutshell, and quantitatively, how the concentrating effect can be regulated. The key is the analyte retention factor that is operating during the introduction of the sample plug into the column. The situation is simple and clear.
To obtain a low value for !!"#$" a large value for the retention factor has to be chosen. The volume ratio !!"#$"/!! expresses the relative volume reduction of the analyte-‐containing solution after it has been applied on the column. This ratio is the inverse of the relative increase in analyte concentration, i.e. the enrichment factor !! !!"#$". The higher retention factor chosen for the sample loading, the smaller becomes the analyte starting zone width/volume, and the higher becomes the enrichment factor.
!!"#$" = !!
1 + !!
!!"#$"!!
= !! ! !!
19
How to use the concentrating effect The width of the analyte starting zone is controlled by the factor 1+ k. The retention factor is as usual governed by the choice of stationary phase (the sorbent) in the column and by the mobile phase, in this case the solvent in the sample plug.
The reason for the concentrating effect is that the analyte migrates very slowly into the column so that it occupies only a very short distance in the early part. This means that the original analyte zone volume and width becomes contracted into a much smaller volume and width, a sample volume contraction. This is obtained simply if the chosen conditions give a very high retention factor since the solvent in the sample works as the mobile phase during sample application.
Useful enrichment can only occur if the analyte is very strongly retained during sample application, perhaps having at least k > 100. During the following elution with k ≈ 0 , as in solid phase extraction, the analytes are released from the stationary phase and eluted in a much smaller volume than the original sample volume.
Even in standard analytical chromatography, where the analyte is dissolved in the same solvent that makes up the eluent (mobile phase), this concentrating effect is significant even if ! = !. Then the analyte concentration increases by a factor of 6 after application to the column top. Similarly, the analyte starting zone width/volume is 6 times smaller than in the injected sample. This helps significantly to keep down the total zone broadening, especially considering that ideal chromatography assumes that the width of the starting zone is negligible compared to the regular zone broadening occurring during the analyte migration through the remainder of the column. Except this, the concentrating effect can be utilized for the following purposes.
A. Application of large sample volumes, relative to the column volume (even of equal size or more as the column volume), on regular chromatography columns. This intends to eliminate the contribution of the otherwise too wide analyte starting zone that would have caused deterioration of the separation efficiency. For example, if a sample solution is 1 ml and the analyte mass is enough for detection after the column, a standard 150 x 4,5 mm HPLC column can be used if the sample solvent is chosen so that it gives a very high retention factor, hence a “weak” solvent. A typical case is a reversed-‐phase HPLC column eluted with a methanol – water (1 + 1) eluent. If the sample is dissolved in pure water a strong concentrating effect will be obtained.
B. Enrichment of the analyte concentration for trace analysis directly on the separation column or a pre-‐column (sample volume >> column volume). Enrichment is necessary if the analyte concentration in the sample is much below the detection limit. To reach an analyte mass above the detection limit it will be necessary to use a huge sample volume and have its analyte concentrated on the top of the column. There are example when 1000 ml sample volumes have been concentrated on standard sized HPLC columns.
20
C. Solid phase extraction. Now, back to the subject of solid phase extraction. In principle the method contains the following two extraction steps (phase transfers) of the analytes that are present in the original water-‐based sample (the liquid):
1. Liquid -‐ solid extraction
2. Solid -‐ liquid extraction
In the liquid-‐solid extraction step the process is equal to that described in the Figure above and the aim is to concentrate the analyte in the short extraction column. This requires strong retention obtained by proper choice of the solvent in the sample. To have an idea of the level of the retention factor during sample introduction makes the choice of conditions more logical than just guessing.
In more detail these steps involve:
1. An extraction of the analyte from the aqueous sample onto the solid phase, hence an adsorption (sorption) from the aqueous phase onto the solid phase.
2. The next step is to elute the analyte from the solid phase by using a suitable solvent, hence a desorption from the solid phase into the solvent.
The analyte recovered in step 2. can next be injected onto an analytical separation column, separated, detected and quantified. There are many other steps and precautions that need to be included such as
1. Wetting and conditioning of the extraction column
2. Sample application to the extraction column (= 1. above)
3. Washing of the extraction column
4. Elution of the analyte from the extraction column (recovery of the analyte) (= 2. above)
1. In the first step the SPE-‐column is wetted by a solvent with good wetting properties. Next the column is conditioned with a solvent of equal elution strength as the aqueous sample solvent. 2. In the second step the sample is applied onto the column packing so that the analytes are very well retained on it. This requires that the solvent in the sample is a very “weak” solvent in terms of elution strength so that the retention factors of the analytes become very high. During the sample application the sample solvent itself will function as the mobile phase. The combination of the stationary phase in the extraction column with the “mobile phase” in the sample solution should be chosen so that k >> 1 for the analytes. 3. After sample application the column is washed with a solvent of equal elution strength as the sample solvent to remove substances that are unretained (! = 0) in order to minimize their presence in the final analyte fraction. 4. In the final step the analyte is eluted in a small volume with a “strong” mobile phase solvent, and either injected directly onto the analytical column for analyte separation or collected for further treatment. For the technique to work properly, the analytes must be very strongly retained during step 2. and very weakly retained (easily eluted) during step 4.
21
The lesson to learn is that to achieve a successful SPE you must know, understand, and estimate in the best possible way what the retention factors are in steps 2. and 4. To have a good idea of these is the key to develop successful conditions for SPE. The reason is that both steps are in themselves really chromatographic retention processes.
Quantitative treatment of liquid-‐liquid extraction In liquid-‐liquid extraction an analyte is distributed between two immiscible liquids as described by the distribution equilibrium or extraction equilibrium. To obtain the two-‐phase system the two liquids need to have very different character in terms of hydrophilicity or lipophilicity. For example one of them may have a very low hydrophilicity so that it has a very low or limited solubility in water. Organic solvents such as hexane, dichloromethane, di-‐ethyl ether, and higher alcohols like octanol are good examples of this. The other can have a low lipophilicity (or high hydrophilicity) such as lower alcohols and water. The differences in character depend on the nature of the functional groups in the solvents. High polarity groups contribute to high hydrophilicity whereas low polarity or zero polarity groups contribute to low hydrophilicity. Simply, it can be understood that the two-‐phase system will occur because of the relative insolubility of the phases in each other.
The typical example of a liquid-‐liquid phase system is the combination of water with for example one of the organic solvents mentioned above so that one phase is rich, or even nearly pure, in water, hence the aqueous phase (aq), and the other is rich in organic solvent, hence the organic phase (org). These systems are often called aqueous/organic liquid extraction.
To use the LLE systems for extraction into the organic phase, and obtain a suitable % fraction extracted, it is necessary to understand and express quantitatively how an analyte will be distributed between the two phases. The first criterion to fulfil is of course that the analyte has enough solubility in the organic phase. The other is that the analyte prefers to go to the organic phase rather than to the aqueous phase. Otherwise it will be difficult to obtain all of the analyte in the organic phase (the extract). This has to do with the relative magnitude of intermolecular interactions between the analyte molecules and the solvent molecules in the respective phases.
The following systematic presentation is relevant for low molar mass solutes, such as most organic compounds, but usually not for high molar mass solutes such as proteins, starch, cellulose and more.
A neutral nonprotic analyte
Distribution constant
Consider a non-‐charged analyte, A, that initially is dissolved in one of the phases in an aqueous/organic two-‐phase system. After dissolution, for example in the aqueous phase, and next adding the organic phase, the mixture will be equilibrated by thorough mixing (shaking;
22
convection). If the analyte can be transferred (extracted, distributed, partitioned) partly to the organic phase and equilibrium has been reached (it is a regular dynamic equilibrium process) it is possible to define an equilibrium reaction (or in this case a distribution equilibrium, see more below) as
!!" ⇋ !!"# equilibrium reaction (distribution equilibrium)
for which the equilibrium constant (in this case the distribution constant, see more below) will be given by
equilibrium constant (distribution constant)
Here the symbol [A] denotes the concentration of species A, meaning only this single molecular form of the substance, that is, only this single species.
WHAT IS A SPECIES? (n. plural species). A defined individual molecular chemical entity.
Since the equilibrium involves two different phases it is sometimes called a two-‐phase equilibrium. The subscripts “org” and “aq” refer to the two liquid phases. In older literature the distribution equilibrium is known as Nernst´s distribution law. For a very long time the distribution constant has been known also under the name “partition coefficient” despite the fact that it is an equilibrium constant. The partition coefficient is still frequently used and whenever you see it you can interpret it as being identical to the distribution constant. However, in the present text the term distribution constant will be used consistently. No matter what, it is always important to clearly define any term and symbol used, otherwise it has no logical meaning.
What determines the value of the distribution constant, KD? The distribution constant would depend on the relative size of the intermolecular forces between the analyte molecules (solutes) and the solvent molecules in each phase. These depends on both physical and chemical intermolecular interactions. Obviously, the most important parameter is the molecular structure of the analyte. The intermolecular interactions of a solute in a given solvent would likely be reflected in the solubility of the analyte in that solvent. Therefore the rule of “like-‐dissolves-‐like” is very helpful in predicting the trends in distribution constants that can occur due to the changes of functional groups. For example, if one analyte is exchanged for another, or if the solvent in the organic phase is changed, the distribution constant would change in some direction (but which?). For example, an increase of the solubility in the organic phase would be expected to contribute to an increase of the distribution constant. It is helpful to discuss the properties of solutes and solvents in terms of their hydrophilicity, hydrophobicity, and lipophilicity.
To understand the relationship between molecular structure and the strengths of intermolecular forces it is a good idea to once again consider your text books in General/Fundamental chemistry/Physical chemistry on this matter. The following well-‐known
aq
orgAD A
AK
][][
)( =
23
kinds of intermolecular forces play a very important role in liquid-‐liquid distribution. They are listed by decreasing order of strength:
The last kind, 4., is rather non-‐selective, since it exists between any pair of organic solutes, but still is important because it may explain that the KD(A) increases with increasing carbon chain length in the analyte. The interaction types 1. – 3. are more selective to molecular structure since they depend on the presence of certain functional groups, namely the polar groups that contain polar bonds. Hence, it is important to understand the character of functional groups in a solute and a solvent and how the solute and solvent can interact with each other.
Hydrogen bonding is the most important. The strength of the hydrogen bond increases with the proton donating (protogenic) property in the following order for some selected compounds:
CHCl3 < alcohols < phenols < thiophenols.
It also increases with the proton-‐accepting (protophilic) property of the counter-‐part in the order:
sulphides < unsaturated hydrocarbons < nitriles < neutral oxygen compounds < amines.
Some simple rules-‐of-‐thumb can be stated regarding the choice of extraction solvent:
Hydrogen accepting solvents (protophilic): ethers, esters, ketones, water. Used when in need to increase DA of hydrogen donating solutes: carboxylic acids.
Hydrogen donating solvents (protogenic, protic): chloroform, dichloromethane, water. Used when in need to increase the DA of hydrogen-‐accepting solutes: amines.
Amphiprotic solvents: higher alcohols (butanol, pentanol, octanol, water). These are both hydrogen accepting and hydrogen donating. Can be used as such or as modifiers in hydrocarbon solvents.
Hydrocarbon solvents (nonpolar, aprotic, non-‐protogenic, non-‐protophilic ): heptane, hexane, decane.
Protophilic (solvent): Capable of acting as proton acceptor, strongly or weakly basic (as a Brønsted base ). Also called HBA (hydrogen bond acceptor) solvent. See also: protogenic solvent. Source: PAC, 1994, 66, 1077 (Glossary of terms used in physical organic chemistry (IUPAC Recommendations 1994)) on page 1153.
1. Hydrogen bonding 2. Dipole – dipole 3. Dipole – induced dipole 4. Induced dipole – induced dipole (other terms: dispersion forces, London forces)
24
Protogenic (solvent): Capable of acting as a proton donor (strongly or weakly acidic as a Brønsted acid). The term is preferred to the synonym protic or the more ambiguous expression acidic by itself. Source: PAC, 1983, 55, 1281 (Glossary of terms used in physical organic chemistry) on page 1348.
A polar bond has a permanent dipole quantified by its dipole moment. A nonpolar bond is a covalent bond between two atoms that has zero dipole moment. A polar molecule has a non-‐zero dipole moment caused by presence of one or more polar bonds. A nonpolar molecule has zero dipole moment due to the presence of only nonpolar bonds. However, a nonpolar molecule actually can contain polar bonds if these are symmetrically distributed. Carbon dioxide and benzene are well-‐known examples. Therefore they can interact with other molecules by the interaction types 1., 2., and 3. above, even if the molecules as a whole strictly are nonpolar. Hence, it is tempting to call them polar molecules but this is not correct. It is therefore better to reason about the presence of polar groups in a molecule.
The term polarity is commonly used to characterise solutes and solvents. Strictly it should reflect only the dipole moment of compounds and the term polarity should therefore be reserved to this, only. However, almost invariably, people talk about polarity and polarity scales in completely different aspects such as ordering solubility of a solute in different organic solvents, ordering the solubility of different solutes in a specific organic solvent, and ordering the retention times of different solutes in liquid chromatography. The following citation from IUPAC illustrates the situation:
What is polarity? When applied to solvents, this rather ill-‐defined term covers their overall solvation capability (solvation power) for solutes (i.e. in chemical equilibria: reactants and products; in reaction rates: reactants and activated complex; in light absorptions: ions or molecules in the ground and excited state), which in turn depends on the action of all possible, nonspecific and specific, intermolecular interactions between solute ions or molecules and solvent molecules, excluding such interactions leading to definite chemical alterations of the ions or molecules of the solute. IUPAC Compendium of Chemical Terminology 1142 of 1622. Source: PAC, 1994, 66, 1077 (Glossary of terms used in physical organic chemistry (IUPAC Recommendations 1994)) on page 1151.
Distribution ratio
There are numerous situations when a compound can exist in several different forms, species, in a solution. Analytically, therefore, it may be more important to consider the distribution (extraction) of all substance forms (all species of the substance), not only one (such as for example the species A above). The main reason is that most quantitative determination methods measure all forms together, since different species cannot be distinguished.
To treat this situation quantitatively it is necessary to introduce a different kind of concentration expression, the total concentration. It is defined as the sum of the concentrations of all present species and is symbolized by capital C. For the
25
substance/compound A (swe. ämne) its total concentration is symbolized by CA. If substance A exists only as one species, A, the total concentration reduces to
!! = !
To express the liquid-‐liquid distribution of compound A, assuming for the moment that it can occur also as other species in each of the phases (for example as dimer, or complexed to a complexing agent if in the organic phase, or as the corresponding base of an acid), it is necessary to define the distribution ratio, DA, as the ratio of the total concentrations of A in the two phases,
which results in
Each total concentration means the sum of the concentrations of each species that is present. In this case compound A exists only as one single species, A, which means that
and
and then
which is equal to the above distribution constant for the species A, KD(A). Hence, when only one single species is present, the distribution ratio will equal the distribution constant:
More about total concentrations
The meaning and use of total concentrations will be further clarified if a case is chosen in which the non-‐charged compound is a monoprotic acid, which can form a dimer in the organic phase but also undergo acid-‐base equilibrium with water in the aqueous phase. This can occur for
DA =TotalconcentrationAorgTotalconcentrationAaq
DA =CA,org
CA,aq
CA,org = A[ ]org
CA,aq = A[ ]aq
DA =[A]org[A]aq
)(ADA KD =
26
certain compounds carrying the highly polar carboxyl groups when dissolved in a hydrophobic solvent like toluene. For benzoic acid, HBa , a dimerization equilibrium can be written as
HBaorg + HBaorg ⇌ (HBa)2,org
Of course, benzoic acid can undergo acid-‐base equilibrium with water in the aqueous phase and then the distribution ratio for benzoic acid will be obtained as
If developed further it will be found that the distribution ratio for HBa will be a function of both the distribution constant, KD(Ba), the dimerization constant, and the acidity constant, Ka(HBa). The dimerization and the acid-‐base equilibrium are regarded as side-‐reactions or side-‐equilibria (to the main equilibrium, which is the two-‐phase distribution equilibrium for HBa). Side-‐equilibria can occur in both phases.
As an illustration of the meaning of the total concentration it is interesting to consider an aqueous solution of a polyprotic acid such as EDTA. EDTA, H4A, has four acidic groups and hence four different Ka values, one for each carboxyl group. When dissolved in water all four carboxyl groups can undergo acid-‐base reaction with water resulting in the four corresponding conjugate bases. Then the total concentration of the substance/compound EDTA can be written as
!!!!,!" = !!! !" + !!!! !" + !!!!! !" + !!!! !" + !!! !"
A neutral weak organic acid HX When an organic acid, HX, having the acidity constant Ka(HX), has been added to the pure aqueous phase, realizing that only its neutral acidic form, the conjugate acid, can be distributed to the organic phase, the distribution ratio of compound HX is written
which is based on the acid-‐base equilibrium between HX and water:
!!!" + !!!!" ⇌ !!"! + !!!!"!
Note that the compound HX is present in the aqueous phase in two forms, the two species HX and X-‐, provided that noticeable acid-‐base reaction has occurred. Note also that in the organic phase only the neutral conjugate acid species is present. This is because the ionic conjugate base X-‐ cannot normally be distributed to an organic phase, like any other ionic species.
Two different equilibrium constants are involved: the distribution constant for HX according to
DHBa =CHBa,org
CHBa,aq
=HBa[ ]org + 2 HBa2[ ]orgHBa[ ]aq + Ba!"# $%aq
DHX =CHX,org
CHX,aq
=[HX]org
[HX]aq +[X!]aq
27
and the acidity constant for HX according to
These can next be substituted into the above distribution ratio expression to give
which shows that the distribution ratio will be governed by the values of the two equilibrium constants and, in addition, the hydronium ion concentration. This expression is highly important since it shows how to work with the liquid-‐liquid distribution of a weak organic acid. Clearly, once the constants are given after choosing a certain neutral acid and the extraction solvent, the distribution ratio can only be regulated by the hydronium ion concentration in the aqueous phase. This is the powerful tool that is available for those who want to manage the liquid-‐liquid distribution of an organic acid. Simply, it is a matter of choosing a suitable pH in the aqueous phase as obtained by the addition of a suitable buffer, for example phosphate buffers.
An excellent way to show the effect of pH is to transform the DHX –equation above so that it becomes a function pH. To do this the equation will be expressed in logarithmic form as
log!!" = log!! !" − log 1 +!!(!")!!
!"
This gives a possibility to plot log DHX vs. pH after further transformation to
log!!" = log!! !" + log1
1 +!!(!")!!
!"
and even further to
log!!" = log!! !" + log1
1 + 10!"!!!!(!")
KD(HX ) =[HX]org[HX]aq
Ka(HX ) =H +!" #$aq X%!" #$aq[HX]aq
DHX =KD(HX )
1+Ka(HX )
[H +]aq
28
This shows how log D will vary with the experimental conditions, that is, the pH in the aqueous phase. A very useful illustration of the situation is obtained if the above log DHX – equation is presented as a graph of log DHX versus pH. Here follows an example.
Diagram 1: Log D-‐pH diagram for the distribution of a weak acidic analyte, HX, between water and organic phase and that has pKa(HX) = 5,0 and a KD(HX) = 300. The intersection of the dashed lines marks the so-‐called system point.
The graph has a characteristic linear horizontal part which means that the distribution ratio asymptotically approaches a constant value when the pH is decreased so that pH << pKa(HX). This is chosen in practice as pH < pKa(HA) -‐ 2. Another characteristic is the linear part having a slope of -‐1 that occurs when pH >> pKa(HX). This is interpreted in practice as pH > pKa(HA) + 2.
In the intermediate region, pH = pKa(HA) ± 2 , the function is nonlinear but will always pass through a point 0.3 log units below the system point, which is at the intersection between the two asymptotes.
Derivation of the log DHX – pH function.
Now, back to the next previous equation. After inserting the acidity constant and the distribution constant used in Diagram 1 the function above reduces to
log!!" = log!! !" + log1
1 + 10!!
and further
-5
-4
-3
-2
-1
0
1
2
3
0 1 2 3 4 5 6 7 8 9 10 11 12 13
pH = pK a
(HX)
log DHX = log KD(HX) log DHX
29
log!!" ≈ log!! !" + log 1 = log !! !"
Hence, the distribution ratio equals the distribution constant according to
log!!" = log!! !"
with an error of only 0.01 log unit. This reflects that the compound HX is practically completely neutral in this low pH range. In other words, the %fraction as conjugate acid is ≥ 99% while the %fraction of the conjugate base X-‐ is ≤ 1%.
By similar reasoning another approximation is reached for the case pH >> pKa(HX), when the acid is nearly completely in the conjugate base form (ionized), to result in
log!!" ≅ log!! !" + !!!(!") − !"
This is obviously a straight line relationship between log DHX and pH having a negative slope -‐1. The intercept is obtained as [log KD(HX) + pKa(HX)]. The construction line for this is linear with slope -‐1. It is an asymptote valid at pH ≥ pKa(HA) + 2.
The logarithmic diagram is intended for use in planning experimental conditions for extraction experiments and for analysing the first experiments to give clues to eventual further fine-‐tuning of conditions. This is good enough for most cases.
A neutral weak organic base B Accordingly, in analogy with the above case of the acid HX , for a neutral basic analyte B, with the acidity constant Ka(HB+) of the conjugate acid, the following equations and diagram are obtained:
!"!!" + !!!!" ⇌ !!" + !!!!"!
The acid-‐base equilibrium for the conjugate acid and base, is here written as a deprotonation of the conjugate acid and the acidity constant will be
!!(!!!) = ! !" !!
!"
!"! !"
(Note: it is much easier and more systematic to only work with acidity constants and forget about basicity constants.) In this case the distribution constant is defined as
Exercise 3 1. What pH is needed to obtain DHX = 100 of the acid in Diagram 1 using equal phase volumes? Ocular reading of the diagram using pencil and paper is satisfactory. 2. What pH is needed to obtain DHX = 0.01 of the acid in Diagram 1? Ocular reading of the diagram using pencil and paper is satisfactory.
30
!!(!) =! !"#
! !!
and then the distribution ratio is written as
Transfer to a logarithmic expression gives
log!! = log!! ! + log1
1 + 10!!! !!! !!"
This function has, like for the acid HX, two asymptotes, which can be worked out analogously to obtain
Asymptote 1. For pH >> pKa(HB+) or specifically pH = pKa(HB+) ≥ 2 it reduces to
log!! ≅ log!! !
in similarity to the derivation for the acid HX.
Asymptote 2. For pH << pKa(HB+) and more specifically pH ≤ pKa(HB+) -‐ 2
the log DB -‐function becomes
log!! ≅ log!! ! + log1
10!!! !!! !!"
since 10!!! !!! !!" ≥ 100
and then further transformation gives
log!! ≅ log!! ! − !!! !!! + !"
which is a straight line with slope +1. This is Asymptote 2.
)(
)(
,
,
][][][][
+
++
+=
+==
HBa
BD
aqaq
org
aqB
orgBB
KH
KHBB
BCC
D1
31
Diagram 2: Log D-‐pH diagram for the distribution of a neutral basic analyte, B, between water and organic phase.
Here, the horizontal part occurs at pH >> pKa(HB+) meaning that in this range the %fraction of the conjugate base B in the aqueous phase is 99% or higher. The corresponding %fraction of the conjugate acid HB+ is therefore 1% or lower.
log!! ≅ log!! !
In the range pH << pKa(HB+) the function is linear with a slope of +1 according to
log!! ≅ log!! ! − !!! !!! + !"
How to use the log D – pH diagrams (log-‐log plots) This is very simple. The only tools needed are paper, pencil, and ruler. No computer is needed, not even a calculator! This can be done anywhere you are, even in the laboratory. Draw the two logarithmic axes, divide them equally in log units, mark the system point, draw the horizontal line up to one pH unit from the system point, draw the sloping line (slope +1 or -‐1) up to one pH unit from the system point, make a rounded connection between the two straight lines so that it passes through a point 0.3 log units under the system point. That’s it!
-5
-4
-3
-2
-1
0
1
2
3
0 1 2 3 4 5 6 7 8 9 10 11 12 13
pH = pK a
(HB+
)
log DB
pH
log DB = log KD(B)
pKa(HB+) = 8,0
log KD(B) = 2,5
Exercise 4
1. What pH is needed to obtain DB = 100 of the base in Diagram 1 using equal phase volumes? Ocular reading of the diagram using pencil and paper is satisfactory.
2. What pH is needed to obtain DB = 0.01 of the base in Diagram 1? Ocular reading of the diagram using pencil and paper is satisfactory.
32
Applications of liquid-‐liquid extraction
Separation of acids and bases by single batch liquid-‐liquid extraction To separate two analytes in one single extraction it is necessary that one of them goes quantitatively to the organic phase and the other quantitatively to the aqueous phase. Clearly, if assuming equal phase volumes, the distribution ratio needs to be 100 and 0.01, respectively, for this to work. In other words, the ratio of the two D-‐values needs to be ≥104. A few examples follow. The graphical method based on the log D-‐pH diagrams is an excellent tool to approach the problem systematically.
Two neutral weak acids, HX and HY In this case the two graphs, one for HX, and one for HY, are plotted in the same diagram. The data needed are the acidity constants and the distribution constants for each acid.
for HX: KD(HX) = 10000, pKa(HX) = 8.0, For HY: KD(HY) = 1000, pKa(HY) = 4.0. Equal phase volumes.
Figure. Separation of two weak acids HX and HY by liquid-‐liquid distribution in a single extraction. The shaded area shows the pH-‐range that can be used for quantitative separation. To do this a suitable buffer is needed in the aqueous. Phosphate buffer would be a good choice. From G. Schill et al., Separation Methods.
A challenge for You! Use any simulation/calculation computer program to simulate the non-‐approximated equation for log D = f(pH). This is the non-‐approximated function:
log!!" = log!!(!") + log !1
1 + 10!"!!!!(!") !
See if you can get the same graphs as shown above. If so you can generate many more cases to learn from.
33
Two neutral weak bases, codein and papaverin In analogy with the extractive separation of the two acids above the same approach can be used for two neutral bases. Here two basic drug substances are considered. One is codeine (I-‐BC), the other papaverin (II-‐BP). The following data are available: for BC, log KD(Bc) = 2.0 and pKa(HBc+) = 6.4, and for Bp, log KD(Bp) = 4.7 and pKa(HBp+) = 6.4. Equal phase volumes.
Figure. Separation of two weak bases by liquid-‐liquid distribution in a single extraction. The shaded area shows the pH-‐range that can be used for quantitative separation. From G. Schill et al., Separation Methods.
The shaded area shows the pH-‐range that can be used for the separation.
LITERATURE G. H. MORRISON AND H. FREISER, SOLVENT EXTRACTION IN ANALYTICAL CHEMISTRY, WILEY, NEW YORK, 1957.
H. IRVING AND R. J. P WILLIAMS, LIQUID-‐LIQUID EXTRACTION, CH. 31, P. 1309 – 1365, IN I. M. KOLTHOFF AND P. J.
ELVING (EDS.), TREATISE IN ANALYTICAL CHEMISTRY, PART I, SECTION C, VOL. 3, INTERSCIENCE, NEW YORK, 1961.
Y. MARCUS, SOLVENT EXTRACTION OF INORGANIC SPECIES IN CHEM. REV. 63 (1963) 139.
J. A. DEAN, CHEMICAL SEPARATION METHODS, VAN NOSTRAND, NEW YORK, 1969
Y. MARCUS AND A. S. KERTES, ION EXCHANGE AND SOLVENT EXTRACTION OF METAL COMPLEXES, WILEY-‐INTERSCIENCE,
NEW YORK, 1969.
A. LEO, C. HANSCH AND D. ELKINS, PARTITION COEFFICIENTS AND THEIR USES, CHEM. REV., 71, 525 (1971).
G. SCHILL ET AL, SEPARATION METHODS FOR DRUGS AND RELATED ORGANIC COMPOUNDS, SECOND ED.
SWEDISH PHARMACEUTICAL PRESS, STOCKHOLM, 1982; ISBN 91 86274-‐01-‐5.
A. MARTÍN-‐ESTEBAN, SAMPLE PREPARATION FOR CHROMATOGRAPHIC ANALYSIS, CH. 5 IN ADVANCES IN CHROMATOGRAPHY,
VOL. 51, EDS. E. GRUSHKA AND N. GRINBERG, CRC PRESS, TAYLOR & FRANCIS, BOCA RATON, 2014.
IUPAC GREEN BOOK
IUPAC ORANGE BOOK
IUPAC GOLD BOOK
IUPAC TERMINOLOGY IN PHYSICAL CHEMISTRY
CHEMICAL PRINCIPLES, COURSE BOOK FOR ÅK 1 GRUNDLÄGGANDE KEMI FÖR B OCH K
KOMPENDIUM TRANSPORTPROCESSER – KAT KOMPENDIUM I TERMODYNAMIK 1 OCH 2, BENGT JÖNSSON