Sample Preparation. Molecular Properties

Transcription

1 Sample Preparaton Molecular Propertes Wherever you see ths symbol, t s mportant to access the on-lne course as there s nteractve materal that cannot be fully shown n ths reference manual.

2 Ams and Objectves Ams and Objectves Ams To ntroduce the concept of functonal group chemstry as a means of targetng separaton mechansms n Lqud and Sold Phase Extracton To hghlght the dfferent types of functonal chemstry and nvestgate varous physco-chemcal prncples assocated wth dfferent functonal groups To nvestgate the effects of functonal chemstry on analyte solublty and analyte nteracton wth Sold Phase Extracton sorbents To nvestgate the concept of pka and show how ph manpulaton can be used to ncrease the selectvty of Sold Phase Extracton Methods To nvestgate the concept of proten bndng related to Sold Phase Extracton Methodology Objectves At the end of ths Secton you should be able to: Defne the prmary characterstcs of Hydrophobc, Polar, Ionc and Chelatng functonal groups Predct the affects of functonal group chemstry on analyte retenton wth varous Sold Phase Extracton Sorbents Manpulate solvent strength n Sold Phase Extracton Protocols n order to affect analyte retenton or eluton Predct the effect of soluton ph on analyte retenton n Sold Phase Extracton Demonstrate an understandng of chelaton and proten bndng as problems wth sold phase extracton mechansms

3 Content Functonal groups 3 Overvew 3 Interactons 5 Hydrophobc or Non-Polar Groups 7 Overvew 7 Hydrophobc Interactons Solublty 8 Hydrophobc Interactons Sorbents 9 Polar Groups 11 Overvew 11 Polar Interactons Solublty 11 Polar Interactons Sorbents 12 Ionc Groups 14 Overvew 14 Hydrogen concentraton ph 16 Ionsaton Constant Ka 17 Ionsaton Constant pka 18 Ionc strength 19 Ionc Interactons Solublty 20 Ionc Interactons Sorbents 22 Chelatng Groups 23 Overvew 23 Chelatng Interactons Solublty 24 Chelatng Interactons Sorbents 25 Proten Bndng 26 Crawford Scentfc 2

4 Functonal groups Overvew All sample preparaton technques employed depend on the specfc molecular propertes of the analytes, and the characterstcs of the sample matrx. As prevously dscussed, the prmary molecular characterstcs of mportance n sample preparaton when usng extracton approaches are molecular solublty, and specfc functonal group propertes of the analytes, solvents, and sorbents nvolved n the technques. Common analytes n dfferng matrces As a matter of convenence, the prmary propertes of molecules that are relevant to sample preparaton are dvded roughly nto four categores: 1. Non-polar characterstcs: Sample contanng non-polar analytes, endogenous protens and other matrx nterferents 2. Polar characterstcs: Sample contanng polar analytes, endogenous protens and other matrx nterferents Crawford Scentfc 3

5 3. Ionc characterstcs: Sample contanng onc analytes, endogenous protens and other matrx nterferents 4. Chelatng characterstcs: Sample contanng chelatng analytes, endogenous protens and other matrx nterferents Each of these categores s dependent on the presence of dfferent elements wthn the analyte molecule, as well as the propertes of solvents and sorbents nvolved n the sample preparaton process. Each of these specfc categores of propertes wll be addressed n detal throughout ths course. To facltate on a molecule have been assessed, t s possble to draw some conclusons about the propertes of the molecule relevant to the sample preparaton process. These propertes wll be nfluenced by functonal group nteractons. The term functonal group nteractons speaks of the manner n whch one functonal group on, for example, an understandng the propertes of a molecule, t s useful to consder the molecular structure from the standpont of ts varous functonal groups A functonal group s a sub-structure wthn the larger overall structure of a molecule. Ths sub-structure often exhbts characterstcs that are sgnfcantly dfferent from the other structural elements surroundng t. To truly understand the propertes of a molecule that are relevant to sample preparaton, t s necessary to assess and temze as many of the dfferent functonal groups wthn a molecule as possble. Each of these dfferent groups wll play a role n determnng the key nteractons of the molecule wth the sample preparaton process and envronment. Crawford Scentfc 4

6 Interactons Once the functonal groups analyte molecule, nteracts wth the functonal groups n the surroundng envronment; n partcular, a solvent or sorbent nvolved n the sample preparaton process. Each prmary functonal group category exhbts ts own respectve and unque functonal group nteractons. It s the medaton of the nteractons wth the surroundng envronment that allows the sample preparaton process to occur. Each of the categores of nteractons s medated n a dfferent manner. To gan a deep understandng of sample preparaton technques, t s mportant to obtan a clear pcture of these categores of nteractons. Another mportant consderaton regardng solublty of an analyte speces (.e., nteracton wth the solvent envronment) s that the overall solublty characterstcs are a composte of the ndvdual functonal group nteractons wth the solvent. In SPE, by contrast, any ndvdual functonal group nteracton wth the sorbent may domnate the stuaton, n spte of other functonal groups. Non-polar (hydrophobc) analyte functonal groups drectly nteract wth non polar solvent molecules Non-polar analyte functonal groups drectly nteract wth non-polar SPE bonded phase Crawford Scentfc 5

7 Polar (hydrophlc) analyte functonal groups drectly nteract wth polar solvent molecules Hghly non-polar (hydrophobc) analytes show very hgh solublty n non-polar solvents Analytes wth mxed functonal chemstry show reduced solublty n the non-polar solvent due to repulson between the polar functonal group and the non-polar solvent molecules Crawford Scentfc 6

8 In Sold Phase Extracton one sngle functonal group can domnate the nteracton wth the sorbent, regardless of any other functonal groups present on the analyte molecule Hydrophobc or Non-Polar Groups Overvew One major category of functonal group relevant to sample preparaton s termed hydrophobc, or non-polar. Hydrophobc groups are fundamentally hydrocarbon n nature, for the most part contanng no heteroatoms. Hydrophobc groups obtan ther prmary property from Van der Waals forces, also called dsperson forces. Van der Waals forces are short-range, energetcally weak nduced atomc dpoles, exhbted by the hydrogen atoms on hydrocarbons. Hydrophobc functonal groups, as the name mples, prefer a non-aqueous envronment; deally, an organc solvent exhbtng smlar non-polar functonal group characterstcs. Non-polar (Van der Waals) forces Short range Crawford Scentfc 7

9 Hydrophobc Interactons Solublty Hydrophobc functonal groups on analyte molecules exhbt nteractons wth solvents or sorbents termed hydrophobc nteractons. These nteractons consst of Van der Waals forces on the analyte molecule nteractng wth Van der Waals forces n the envronment. These are attractve forces. Lke dssolves lke hydrophobc analyte undergoes ntermolecular Van der Waals nteractons wth hydrophobc solvent From a solublty perspectve, hydrophobc nteractons nfluence analyte molecules to prefer solvent envronments that are smlarly hydrophobc or non-polar n nature. Thus, analytes contanng predomnantly hydrophobc groups wll tend to be soluble n less-polar organc solvents, such as acetontrle, THF, ethyl acetate, chloroform, etc. Such analyte molecules wll be less soluble n hghly aqueous envronments such as water, buffers, or aqueous mxtures contanng low concentratons of organc solvents. Common solvents used to dssolve / elute hydrophobc analytes Crawford Scentfc 8

10 Hydrophobc Interactons Sorbents Due to the attractve Van der Waals forces, hydrophobc groups on analyte molecules wll favor chromatographc nteractons wth extracton sorbents bearng smlar hydrophobc or non-polar groups. Common examples of extracton sorbents exhbtng hydrophobc nteractons nclude C 18, C 8, phenyl, cyclohexyl, and many non-polar polymer sorbents. These sorbents all contan functonal groups wth sgnfcant hydrophobc (hydrocarbon) content. Typcal Hydrophobc Sorbents Hydrophobc analyte-sorbent nteractons are facltated by solvent envronments of low hydrophobcty, n partcular water, buffers, and aqueous mxtures contanng only a very low content of water-mscble organc solvents such as methanol, acetontrle, THF, other alcohols, etc. Snce such solvent envronments exhbt low hydrophobc nteractons, they are ncapable of dsruptng the attractve forces between the analyte and the sorbent. Hydrophobc nteractons Crawford Scentfc 9

11 Hydrophobc nteractons between analyte and sorbent are promoted by the use of low hydrophobcty solvent systems. These solvent systems are usually hghly aqueous and contan only small amounts of water mscble organc solvents. They have a poor affnty for the non-polar functonal groups of the analyte molecule and are therefore unable to dsrupt the analyte-sorbent nteracton. Conversely, hydrophobc nteractons between analytes and sorbents are readly dsrupted by solvents contanng sgnfcant hydrophobc character. Examples nclude most pure organc solvents, such as methanol, acetontrle, THF, ethyl acetate, MTBE, chloroform, hexane, etc. Conversely solvent systems that have a hgh degree of hydrophobcty can be used to dsrupt the analyte-sorbent nteracton. Solvents used to dsrupt hydrophobc analytesorbent nteractons nclude pure organc solvents such as methanol, acetontrle, THF, ethyl acetate, MTBE, chloroform, and hexane. Crawford Scentfc 10

12 Polar Groups Overvew Another major category of functonal group relevant to sample preparaton s termed polar. Polar groups contan dpoles molecular bonds where the electrons n the bond are shared unequally, due to a greater electronegatvty n one of the atoms n the bond versus the other. Therefore, wthn each dpole, one of the atoms s more postve and the other s more negatve, n a sense creatng a molecular magnet. The greater the dfference n electronegatvty between the two groups, the stronger the dpole. Polar Functonal Groups Most common strongly polar groups contan at least a sngle heteroatom such as oxygen, ntrogen, sulfur or phosphorous, often bonded to a carbon atom. Other, weaker common dpoles nclude unsaturated carbon-carbon bonds, aromatc rngs and halogen-carbon bonds. Polar Interactons Solublty Polar functonal groups on analyte molecules exhbt nteractons wth solvents or sorbents termed polar nteractons. In pont of fact, the term polar nteractons encompasses a wde range of dfferent nteracton categores, ncludng dpole-dpole, hydrogen bondng, p-p nteractons, etc. These nteractons consst of a dpole on the analyte molecule nteractng wth a dpole n the envronment. These are attractve forces. Hydrogen bondng (dpole-dpole nteracton) between the solvent (water) and polar groups on the analyte molecule Crawford Scentfc 11

13 From a solublty perspectve, polar nteractons nfluence analyte molecules to prefer solvent envronments that are hghly polar n nature. Thus, analytes contanng predomnantly polar groups wll tend to be soluble n hghly aqueous solvents, ncludng pure water, most buffers, and solutons contanng water or buffers n combnaton wth water-mscble organc solvents at relatvely low concentraton. Such analyte molecules wll be less soluble n more non-polar envronments, such as pure organc solvents ncludng acetontrle, THF, ethyl acetate, chloroform, etc. Typcal Polar Solvents Polar Interactons Sorbents Due to the attracton between dpoles, polar groups on analyte molecules wll favor chromatographc nteractons wth extracton sorbents bearng polar groups on the surface. Common examples of extracton sorbents exhbtng polar nteractons nclude unbonded slca, dol, amnopropyl, cyano, and certan polar polymer sorbents. Also, most slcabased sorbents exhbt polar nteractons va unbonded slanol groups remanng on the surface of the sorbent. Typcal Polar solvents Crawford Scentfc 12

14 Polar analyte-sorbent nteractons are facltated by solvent envronments of low polarty. Examples nclude most pure organc solvents wth mnmal polar character such as acetontrle, THF, ethyl acetate, MTBE, chloroform, hexane, etc. Snce such solvent envronments exhbt low polarty, they are ncapable of dsruptng the attractve forces between the analyte and the sorbent. Hydrophlc (polar) analytes nteractng wth hydrophlc sorbents n a hydrophobc (nonpolar) solvent envronment Conversely, polar nteractons between analytes and sorbents are readly dsrupted by hgh-polarty solvents, ncludng water, buffers, and aqueous mxtures contanng a low content of water-mscble organc solvent. In addton, most alcohols wll dsrupt polar nteractons due to the presence of the hghly polar hydroxyl group on the alcohol. Hydrophlc (polar) analyte nteracton dsrupted by solvent wth hydrophlc character Crawford Scentfc 13

15 Ionc groups Overvew Ionc functonal groups are those whch are capable of donatng a proton (acds), or acceptng a proton (bases). Ionc groups can exst n ether a neutral or an onzed state, dependng on the ph of the envronment. The ph at whch the respectve group becomes neutral or charged s a functon of the specfc characterstcs of the functonal group. Snce the charge state (neutral or onzed) has a huge nfluence on the propertes of the functonal group, t s mportant to be aware of how the partcular functonal group s affected by ph. Acdc group H R N + H H R N H H R O OH R O O Basc groups Low ph Hgh ph As the soluton ph s lowered ths organc acd becomes ncreasngly less onsed and therefore less polar. The pka value represents the ph at whch 50% of the molecules are n an onsed state. Crawford Scentfc 14

16 As the soluton ph s rased ths organc base becomes ncreasngly less onsed and therefore less polar. The pka value represents the ph at whch 50% of the molecules are n an onsed state. Ionc functonal groups exst n conjuncton wth an on of the opposte charge to the organc onc functonal group. Ths on s referred to as the counter-on, and has sgnfcant mplcatons for sample preparaton, n partcular SPE. For example, an onzed acd (negatve charge) may have an ammonum or potassum counter-on (postve charge). Smlarly, an onzed base (postve charge) may have a chlorde or acetate counter-on (negatve charge). Counter ons of onc organc speces can be replaced by counter ons wth a hgher affnty for the onc speces. The table of relatve counter on affntes may be used to consder the relatve bndng strength of counter ons for both analyte ons and sorbent speces. Crawford Scentfc 15

17 Hydrogen Concentraton ph ph s defned as the negatve log of the hydrogen on concentraton n an aqueous soluton. Pure water has a ph of 7, wth a hydrogen on concentraton of 10-7 moles per lter. Snce ph s a log functon, a sngle ph unt change represents a ten-fold (sngle order of magntude) change n the hydrogen on concentraton. Remember: Pure water has a ph of 7, wth a hydrogen on concentraton of 10-7 M. Snce ph s a logarthmc functon, a sngle ph unt change represents a tenfold (sngle order of magntude) change n the hydrogen on concentraton. ph = -Log [H + ] ph of a soluton can be nfluenced by the addton of an acd or a base, both of whch change the hydrogen on concentraton. In partcular, addton of acds, whch are proton donors, wll ncrease the hydrogen on concentraton, thus lowerng the ph. Addton of Crawford Scentfc 16

18 bases, whch are proton acceptors, wll reduce the hydrogen on concentraton, thus rasng the ph. The ph of a soluton wll nfluence the charge state of an acd or basc analyte. For example, addton of an acd to an aqueous soluton of an acdc analyte wll decrease (or suppress) the concentraton of charged analyte n soluton, as the hydrogen on concentraton ncreases. Conversely, rasng the ph by addton of a base wll ncrease the concentraton of the charged form of the acdc analyte. Addton of an acd lowers ph by donatng H + (protons) to soluton. Addton of a base promotes onsaton of the acd analyte. Ionzaton Constant Ka All acds and bases have a unque onzaton constant (Ka), whch specfes the degree to whch the speces onzes n aqueous soluton. The greater ths onzaton, the greater the nfluence of the speces on the hydrogen on concentraton, and thus the stronger the acd or base. For example, a strong acd wll completely onze, creatng a hgh hydrogen on concentraton, and a very low ph n the soluton. Conversely, a strong base wll completely onze by pullng hydrogen ons from the soluton, thus lowerng the hydrogen on concentraton and rasng the ph of the soluton to a very hgh value. Weaker acds and bases wll onze to a lesser degree and therefore have less effect on changng the ph. Crawford Scentfc 17

19 Strong acd - almost fully onsed Weak acd - only partally onsed Strong base most H + ons removed from soluton Weak base most H + ons stll n soluton Ionsaton Constant - pka pka s defned as the negatve log of the onzaton constant (Ka) for a partcular speces. By defnton, a speces n soluton at a ph equal to ts pka wll exst 50% n the neutral state and 50% n the onzed state. When workng wth an onc speces n soluton, t s desrable that all molecules of that speces exhbt the same charge state, snce the propertes of the charged state versus the neutral state can be radcally dfferent. The corollary to ths s that workng at a ph equal to the pka of any gven speces s the worst possble ph to work at, snce 50% of the molecules wll behave n one way, and 50% n another way. Crawford Scentfc 18

20 An organc acd at pka (soluton ph = analyte pka) In order to brng the onc functonal group to ether an essentally complete charged state or an essentally complete neutral state, t s recommended to work at a ph at least two ph unts away from the pka of the speces. As an example, for an acd wth a pka of 4.5, to fully onze the acd the soluton ph should be 6.5 or hgher. For a base wth a pka of 9.0, to fully onze the base the soluton ph should be 7.0 or lower. Ionc strength Another parameter relevant to onc analytes n soluton s referred to as the onc strength of the soluton. Ths s the concentraton of total onc speces n soluton. For example, pure water has a self-onzaton constant of 10-7, yeldng an onc strength of 10-7 M for the hydrogen on and hydroxde on content. Ionc strength of a soluton can be altered by addng acds, bases, or neutral salts to the soluton. In most cases, the onc strength component contrbuted by the self-onzaton of water (10-7 M) s nsgnfcant compared to the contrbuton of the added speces. So for example, f 0.5 moles of NaCl s added to one ltre of pure water, the resultng soluton s sad to be 0.5M n onc strength. Crawford Scentfc 19

21 Ionc strength of a soluton s partcularly mportant n SPE of onc speces. Ionc Interactons Solublty Ionc functonal groups on analyte molecules are very dfferent than hydrophobc or polar groups n that onc group propertes are a functon of the charge state of the speces. Therefore, the nteractons of an onc analyte wth ether a solvent or sorbent envronment wll smlarly be nfluenced by the charge state. From a solublty perspectve, onzed groups tend to favour solublty n hghly polar solvents. Therefore, bases at low ph (onzed) and acds at hgh ph (onzed) wll be more soluble n hghly aqueous solvents, ncludng pure water, most buffers, and solutons contanng water or buffers n combnaton wth water-mscble organc solvents at relatvely low concentraton. Crawford Scentfc 20

22 Solublty of organc acd speces at low ph values. Solublty of organc acd speces at hgh ph values. Solublty basc speces at low ph values. Solublty basc speces at hgh ph values. Conversely, onc groups n a neutral state tend to favor solublty n non-polar solvents. Therefore, bases at hgh ph (neutral) and acds at low ph (neutral) wll be more soluble n more non-polar solvents such as acetontrle, THF, ethyl acetate, chloroform, etc. Crawford Scentfc 21

23 Ionc Interactons Sorbents Ionc functonal groups nteract wth sorbents contanng onc functonal groups of the opposte charge. Therefore, acd analytes (wth a negatve charge) wll nteract wth sorbents contanng basc functonal groups (wth a postve charge). Smlarly, basc analytes (wth a postve charge) wll nteract wth sorbents contanng acdc functonal groups (wth a negatve charge). It s essental, for an effectve nteracton to occur between an onc analyte group and an onc sorbent, that both speces exst n the charged state. Therefore, the ph of the soluton must be approprate. When lookng at the ph to consder onzaton of the analyte and sorbent, t s not mportant whether the analyte s the acd and the sorbent s the base or vce versa. What s essental s that the ph value s correct relatve to the respectve pka values of the analyte and the sorbent. As the ph s rased the sorbent becomes ncreasngly onsed; allowng the sorbent analyte nteracton to occur. Between a ph of 6.5 and 8 all analyte and all sorbent speces are onsed ant therefore maxmum analyte-sorbent nteractons are occurrng. (the upper part of the fgure presents the behavour at low ph values, the lower part presents behavour at hgh ph values). Specfcally, as prevously stated, the ph should be at least two ph unts below the pka of the base, and, smultaneously, at least two ph unts above the pka of the acd. Wth a base havng a pka of 10.0 and an acd havng a pka of 4.5, ths condton would be met at a ph anywhere from 6.5 to 8.0. Crawford Scentfc 22

24 The 2 ph rule plays an mportant part n analyte behavour durng sold phase extracton. Here we see an acdc analyte that does not nteract wth the basc sorbent surface untl t s charged (the upper part of the fgure presents the behavour at hgh ph values, the lower part presents behavour at low ph values). Chelatng Groups Overvew A category of functonal group that s often overlooked n sample preparaton s that of chelatng functonal groups. Chelatng groups have a hgh affnty for transton metals, and are often combnatons of common polar or onc groups that together exhbt hgh bndng energes for the metals n queston. Examples of chelatng groups nclude amnes, multple amnes, multple carboxylc acds, or acds n combnaton wth amnes. Chelatng process Crawford Scentfc 23

25 Chelatng Interactons Solublty Chelatng functonal groups contrbute to solublty characterstcs of ther respectve analyte molecules n the same way as the composte of the ndvdual functonal groups consttutng the chelatng group. For example, a chelatng group composed of two amnes and a carboxylc acd wll affect solublty n the same way as the ndvdual two amnes and acd. Snce chelatng groups often contan ndvdual onzable groups, the mpact on solublty can also be nfluenced by ph. Amne groups wthn the chelatng group wll favour solublty n organc solvents at hgh ph (neutral charge), and favor solublty n polar solvents at low ph (postve charge). Smlarly, carboxylc acd groups wthn the chelatng group wll favor solublty n organc solvents at low ph (neutral charge), and favor solublty n polar solvents at hgh ph (negatve charge). Alpha keto glutarate solublty at low ph values Alpha keto glutarate solublty at medum ph values Alpha keto glutarate solublty at hgh ph values Crawford Scentfc 24

26 Chelatng Interactons Sorbents Chelatng groups may be used to effect hghly selectve and unque extractons when performng SPE, through n-stu modfcaton of commercally-avalable sorbents wth certan metals. Another mportant mplcaton of chelatng groups s ther nteracton wth resdual metals n commercal sorbents. Durng the manufacturng process, t s vrtually mpossble to create metal-free sorbents n SPE, due to the hgh cost of ths process. Therefore, all commercal SPE-grade sorbents avalable have some content of resdual metals, whch can create problems durng sample preparaton f overlooked as a retentve presence. Due to the unque nature and hgh energy of the chelatng nteracton, once establshed (for example, retenton on an extracton sorbent), the nteracton requres strong approaches for dsrupton prmarly the addton of compettve chelatng agents, or the addton of strong acds or bases. The alpha keto glutarate molecule can only be dsplaced through the use of strong acds to on-supress the analyte speces -dsruptng the analyte/sorbent nteracton Chelatng groups can be used to perform hghly selectve extracton procedures through n-stu nteractons wth transton metals contaned n the base slca. Ths chelatng nteracton can be dffcult to suppress due to ts hghly energetc nature. Crawford Scentfc 25

27 Here we see a completng chelatng speces n soluton dsruptng the analyte/sorbent nteracton. Now that we have revewed molecular propertes va functonal groups, we are almost ready to look n greater detal at the most common sample preparaton approaches. Before we do however, there s one addtonal topc to address proten bndng. Proten Bndng Proten bndng refers to the affnty of many chemcal compounds for nteracton wth natve protens n the orgnal sample. In pharmaceutcal applcatons, proten bndng s qute common, snce many aspects of pharmaceutcal chemstry occur through drug nteractons wth protens, ncludng transport, metabolsm and target effcacy. However, proten bndng of an analyte can often lead to problems durng extracton procedures f the proten- bound analyte s not frst freed from the proten nteracton, snce the analyte wll behave as part of an analyte/proten complex, rather than as an analyte free n soluton. Crawford Scentfc 26

28 Here, the analyte molecules may become attached to the proten structure whch wll mean durng sample preparaton that the analyte behaves as that analyte/proten complex rather than the free analyte n soluton. Most commercal SPE materals are desgned wth the greatest surface area possble. Ths hgh surface area allows for sorpton of the greatest possble mass of analyte per mass of sorbent. Most hgh surface sorbents have commensurately small pore sze. Small chromatographc pores can be a beneft n that they wll exclude from retenton many large contamnant molecules, n partcular protens, whch often cause major problems n the analyss. Unfortunately, f a desred analyte s bound to an excluded proten, the analyte may be carred through the extracton column unretaned, along wth the excluded proten. If the analyte s bound to the proten, then t s prevented from nteractng correctly wth the SPE sorbent as t s effectvely excluded from the pores wthn the sorbent. Ths wll result n very poor analyte retenton. Therefore, t s mportant that proten-bound drugs frst be released or unbound from the proten pror to applcaton of the sample to the sorbent column. Ths can be accomplshed n a varety of ways, ncludng ph changes, addton of organc solvents or detergents, or the use of chaotropc agents, whch dsrupt the three-dmensonal structure of the proten. Once the analyte s released from the proten, normal analyte-sorbent nteractons can occur. Ths s often equally mportant for lqud/lqud extracton as well. Crawford Scentfc 27

29