Sample Preparation. Solid Phase Extraction Mechanisms

Transcription

1 Sample Preparaton Sold Phase Extracton Mechansms 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 hghlght the varous steps n the Protocol for varous Modes (Sorbents) for conventonal sold phase extracton procedures ncludng: o Non-polar SPE o Polar SPE o Caton-Exchane SPE o Anon-Exchange SPE o Mxed mode SPE For each mode the varous portocol steps wll be examned n terms of the solvents and solutons used, sorbents used, solvent addtves, ph and onc strength manpulaton Objectves At the end of ths Secton you should be able to: Identfy the correct SPE mode to optmse the selectvty of the procedure for the analyte of nterest Choose the correct sorbent for varous modes of SPE Optmse analyte retenton through correct sorbent equlbraton, condtonng and sample loadng by controllng sorbent actvty, sample and sorbent onc strength, analyte and sorbent degree of onsaton Maxmse extract cleanlness by optmsng the wash soluton solvent strength and onc strength and ph where approprate Maxmse analyte recovery and selectvty by optmsng the eluton solvent and onc strength and ph where approprate

3 Content Non-polar SPE 3 Overvew 3 Sample Pretreatment 5 Equlbraton and Sample Loadng 9 Washng and Analyte Eluton 12 Polar SPE 14 Overvew 14 Sample Pretreatment 17 Sorbent Condtonng, Equlbraton and Sample Loadng 18 Sorbent Washng and Analyte Eluton 19 Caton-Exchange SPE 21 Overvew 21 Sample Pretreatment 25 Sorbent Condtonng, Equlbraton and Sample Loadng 26 Washng and Analyte Eluton 28 Anon-Exchange SPE 30 Overvew 30 Sample Pretreatment 33 Sorbent Condtonng, Equlbraton and Sample Loadng 34 Washng and Analyte Eluton 35 Mnmum pka Dfferental 37 Mxed-Mode SPE 40 Overvew 40 Sample Pretreatment 44 Sorbent Condtonng, Equlbraton and Sample Loadng 45 Washng and Analyte Eluton 46 Summary 49 Crawford Scentfc 2

4 Non-polar SPE Overvew Non-polar SPE s the extracton process where molecules wth non-polar functonal groups are extracted from predomnantly polar matrces onto sorbents exhbtng non-polar surface propertes. In non-polar SPE, the retenton mechansm s the nteracton of non-polar groups on the analytes of nterest and the non-polar functonal groups on the sorbent, va Van der Waals forces. Ths nteracton s dsrupted (causng eluton of the analytes) by solvents wth sgnfcant non-polar character. Ths nteracton s facltated (allowng retenton of the analytes) by solvents havng very lttle non-polar character; or, n other words, very polar solvents. Loadng: Column washed wth a polar solvent Snce water s the most polar solvent of all, non-polar SPE s an deal technque for extracton of aqueous samples. Snce most pharmaceutcal samples encountered n boanalyss are aqueous, non-polar SPE s wdely used. Crawford Scentfc 3

5 Eluton: Column washed wth a non polar solvent Examples of common sorbents used n non-polar SPE nclude C18, C8, C6, C4, C2, phenyl, cyclohexyl, and propylcyano. These surface chemstres are most often bonded to a base of slca gel. In addton, many commercal polymers also exst that are fundamentally non-polar n nature. Sorbents Crawford Scentfc 4

6 Sample Pretreatment There are sx basc steps n non-polar SPE, as n most SPE procedures. These steps are sample pretreatment, sorbent condtonng, sorbent equlbraton, sample loadng, washng, and analyte eluton. Sample pretreatment Sample pretreatment of aqueous samples for non-polar SPE typcally nvolves sample dluton, ph adjustment/control, dsrupton of proten bndng, and possbly fltraton f the sample contans partculate matter. Crawford Scentfc 5

7 In general, aqueous samples such as plasma or serum should be dluted at least one-toone wth an aqueous buffer. Dluton reduces the vscosty of the aqueous sample, mprovng chromatographc mass transfer, and thus mprovng the effcency of the extracton. Dluton of plasma sample Crawford Scentfc 6

8 Poor analyte retenton ph adjustment may be necessary for certan onzable speces. For onzable analytes whose polarty changes dramatcally as a functon of ther charge, the sample ph should be adjusted to neutralze the analytes, thus ncreasng ther hydrophobcty, and mprovng retenton on a non-polar sorbent. Good analyte retenton Crawford Scentfc 7

9 Proten bndng, f actve, must be dsrupted so the analytes are free n soluton to partton onto the sorbent surface. Ths dsrupton may be mplemented by addton of an organc solvent (such as methanol), addton of chaotropc agents lke urea or guandne, or a ph adjustment. Proten bndng Proten bndng dsrupton by organc solvent addton Crawford Scentfc 8

10 Fnally f large amounts of partculates are present, t may be necessary to flter the samples. For smaller amounts of partculates, the SPE column frt may do an adequate job of fltraton. Equlbraton and Sample Loadng Fltraton process Many non-polar SPE sorbents are not water-wettable. For such sorbents, analytes appled n an aqueous sample wll not partton onto the sorbent, due to lack of a proper phase nterface between the dry sorbent surface and the sample. Condtonng wth a water-mscble organc solvent such as methanol, acetontrle or tetrahydrofuran wets the sorbent surface and allows the analytes to break through the surface tenson of the water and move nto the sorbent. Poor wettng of surface lttle or no analyte retenton Crawford Scentfc 9

11 Column condtonng Just pror to applyng the aqueous sample, the condtonng solvent should be dsplaced from the sorbent usng an equlbraton solvent. The equlbraton solvent also prepares the sorbent surface to receve the sample. In order that the chemstry of the sorbent remans constant throughout the sample loadng step, the equlbraton solvent should be as smlar to the composton of the sample as possble. For aqueous samples, n most cases, ths means equlbratng the sorbent wth the same buffer as used to dlute the sample. Column equlbraton Good surface wettng and equlbrum analyte retenton Crawford Scentfc 10

12 Durng sample loadng, the sngle most mportant consderaton s the speed wth whch the sample s appled. Too rapd an applcaton wll not allow suffcent tme for the analytes to partton nto the sorbent surface, resultng n analyte breakthrough and poor recovery. Analyte velocty too hgh to nteract wth sorbent functonal groups As a reference, for a 100 mllgram bed mass, a reasonable sample loadng flow rate for non-polar SPE s no faster than a few mlllters per mnute. Optmum analyte velocty enables nteracton wth sorbent functonal groups Crawford Scentfc 11

13 Washng and Analyte Eluton The non-polar retenton mechansm s facltated by polar solvents (for example, water) and dsrupted by solvents contanng some non-polar character (methanol, acetontrle, sopropanol, tetrahydrofuran, and even more non-polar organcs). Solvent washng and analyte eluton Crawford Scentfc 12

14 Therefore, n order to elute frst nterferences, then analytes from the surface, t s necessary to apply solvents of ncreasng hydrophobcty to the sorbent bed. Ths s accomplshed by addng the above organcs to the buffer n ever hgher concentratons. Important: Organc solvents are added to the buffer system to alter nonpolar retenton. A common selecton of organc solvents s: 1. Water 2. Methanol 3. Acetontrle 4. Isopropanol 5. Tetrahydrofuran (THF) 6. Chloroform The optmum wash solvent to use for non-polar extracton s a solvent that s non-polar enough to elute as many nterferences as possble, but not suffcently non-polar to elute the analytes. The best way to dentfy such a solvent s to wash the surface startng wth the equlbraton buffer, then use progressvely more non-polar solvents; for example, frst 100% buffer, then 90% buffer / 10% organc, then 80% buffer / 20% organc, etc. Each wash step should be collected and analyzed for presence of analytes. The wash contanng the hghest percentage organc but no analytes wll provde the cleanest fnal extract. Buffer composton Crawford Scentfc 13

15 For eluton, t s best to use the weakest eluton solvent that has just enough hydrophobcty to gve quanttatve analyte eluton. Such a solvent wll leave behnd on the sorbent surface the maxmum number of contamnants. Too strong an eluton solvent wll brng these addtonal contamnants off wth the analytes. Ths solvent may be dentfed n the same experment used to dentfy the optmum wash solvent. Polar SPE Overvew Polar SPE s the extracton process where molecules wth polar functonal groups are extracted from predomnantly non-polar matrces onto sorbents exhbtng polar surface propertes. Polar nteractons n a non-polar matrx Crawford Scentfc 14

16 Polar nteractons dsrupted by a polar solvent In polar SPE, the retenton mechansm s the nteracton of polar groups on the analytes of nterest and polar functonal groups on the sorbent, typcally va dpole-dpole or hydrogenbondng nteractons. These nteractons are dsrupted (causng eluton of the analytes) by solvents wth sgnfcant polar character (.e., contanng dpoles and/or heteroatoms, especally polar oxygen or ntrogen atoms). Ths nteracton s facltated (allowng retenton of the analytes) by solvents havng very lttle polar character; or, n other words, very non-polar solvents. Most pharmaceutcal bologcal samples are aqueous n nature. Water s a very poor solvent n whch to begn a polar SPE protocol, snce by defnton water s extremely polar, makng t a very strong eluton solvent for polar extractons. Therefore, analytes appled to polar SPE columns drectly from aqueous matrces tend to breakthrough the sorbent bed. An deal ntal step for polar SPE of bologcal samples s a lqud/lqud extracton nto an approprate non-polar solvent. Crawford Scentfc 15

17 Sample appled n aqueous matrx -analyte breakthrough occurs Sample appled n organc matrx -good analyte retenton on sorbent Aqueous versus organc matrx retenton Examples of common sorbents used n polar SPE nclude dol, amnopropyl, propylcyano, unbonded slca, alumna and Florsl. Less common but nevertheless qute useful are on-exchangers, whch do not functon as onc sorbents n non-polar solvent envronments, but often have consderable polar character n a non-polar organc solvent. Other than alumna and Florsl, most phases used for polar extractons are slca-based polymer-based polar sorbents are rare. Common sorbents Crawford Scentfc 16

18 Sample Pretreatment There are sx basc steps n polar SPE, as n most SPE procedures. These steps are sample pretreatment, sorbent condtonng, sorbent equlbraton, sample loadng, washng, and analyte eluton. However, the sorbent condtonng and sorbent equlbraton steps n polar SPE are often combned nto a sngle step. Sample pretreatment of aqueous samples for polar SPE typcally nvolves transferrng the analytes nto a non-polar organc solvent, va a lqud/lqud extracton. Ths s necessary snce the orgnal aqueous sample matrx wll generally cause analyte breakthrough f appled drectly to the polar SPE column. As n any lqud/lqud extracton, t may be necessary to adjust the ph of the orgnal sample to facltate transfer nto the organc solvent. For example, f the analytes are onc, the ph of the aqueous sample should be adjusted to a value that wll convert the analytes to the neutral (uncharged) form, as ths wll enhance solublty n the non-polar organc. It may also be approprate to flter the orgnal sample to remove partculates that are present. Sometmes the ntal lqud/lqud extracton wll cause protens n the sample to precptate, and these should be removed pror to sample applcaton to the SPE column. Proten bndng of analytes s less of a concern n ths process than n non-polar SPE, snce the ntal lqud. Fltraton process Note - When performng lqud/lqud extracton as the frst step of a protocol pror to polar SPE, t s not necessary to be greatly concerned as to the qualty of the extract. Instead, the focus should smply be on quanttatve transfer of the analyte nto the non-polar organc. Unlke the process whereby lqud/lqud extracton s the prmary overall sample preparaton procedure, n ths case the polar SPE process wll provde the necessary selectvty n the sample cleanup. Crawford Scentfc 17

19 Clean quanttatve analyte extracton Low selectve quanttatve analyte extracton SPE step provdes hgh selectvty Sorbent Condtonng, Equlbraton and Sample Loadng Sorbent condtonng and equlbraton for polar SPE s usually qute smple. In most cases, the sorbent bed s prepared by treatment wth a few column volumes of a non-polar organc solvent; n many cases ths s the same solvent nto whch the analytes have been extracted from the orgnal aqueous sample. So, for example, f the orgnal sample has been extracted wth hexane, transferrng the analytes nto the hexane layer, then the sorbent bed should be condtoned wth a few column volumes of pure hexane. In most cases t s not necessary to equlbrate the sorbent as an addtonal step pror to applyng the hexane extract of the orgnal sample. Durng sample loadng, the sngle most mportant consderaton s the speed wth whch the sample s appled. Too rapd an applcaton wll not allow suffcent tme for the analytes to partton nto the sorbent surface, resultng n analyte breakthrough and poor recovery. Analyte velocty too hgh to nteract wth sorbent functonal groups Crawford Scentfc 18

20 For polar SPE, the sample applcaton rate should be slower than for a typcal non-polar SPE procedure. Ths s necessary n order for the analyte polar groups to orent n space as they pass through the sorbent bed, such that these groups come n close proxmty to polar sorbent groups, thus allowng a retentve nteracton to occur. Optmum analyte velocty enables nteracton wth sorbent functonal groups Sorbent Washng and Analyte Eluton The polar retenton mechansm s facltated by non-polar solvents (for example, hexane, chloroform, MTBE) and dsrupted by solvents contanng some polar character (THF, ethyl acetate, sopropanol, acetontrle, methanol, water and combnatons of water-mscble organcs wth water). Therefore, n order to elute frst nterferences, then analytes from the surface, t s necessary to apply solvents of ncreasng hydrophlcty to the sorbent bed. Ths s accomplshed by movng from the frst group of solvents to the second group mentoned above. Srong analyte sorbent nteracton n non-polar solvent Crawford Scentfc 19

21 Polar nteractons dsrupted by a polar solvent The optmum wash solvent to use for polar extracton s a solvent that s polar enough to elute as many nterferences as possble, but not suffcently polar to elute the analytes. The best way to dentfy such a solvent s to wash the surface startng wth the solvent, then use progressvely more polar solvents; for example, f the sample extract was n chloroform, frst wash wth 100% chloroform, then 90% chloroform/10% methanol, then 80% chloroform/20% methanol, etc. Each wash step should be collected and analyzed for presence of analytes. The wash contanng the hghest percentage organc but no analytes wll provde the cleanest fnal extract. For eluton, t s best to use the weakest eluton solvent that has just enough hydrophlcty to gve quanttatve analyte eluton. Such a solvent wll leave behnd on the sorbent surface the maxmum number of contamnants. Too strong an eluton solvent wll brng these addtonal contamnants off wth the analytes. Ths solvent may be dentfed n the same experment used to dentfy the optmum wash solvent. Crawford Scentfc 20

22 Caton-Exchange SPE Overvew Caton-exchange SPE s the extracton process where molecules wth basc (catonc) functonal groups are extracted from predomnantly aqueous matrces onto sorbents wth acdc (anonc) surface groups. Catonc analyte nteractng wth an onc sorbent In caton-exchange SPE, the retenton mechansm s the nteracton of charged, catonc groups on the analytes of nterest and charged, anonc functonal groups on the sorbent, va electrostatc (onc) nteractons. These nteractons are dsrupted (causng eluton of the analytes) by one of three prmary methods: a) hgh onc strength, n whch the presence of large numbers of ons n the eluton buffer creates competton wth the analyte groups for the sorbent groups Crawford Scentfc 21

23 Analyte/sorbent nteracton dsrupted by hgh onc concentraton solvent b) a ph change, n whch ether the charged analyte groups are neutralzed, or the charged sorbent groups are neutralzed Charge neutralsaton of the sorbent surface causes eluton of the analyte Crawford Scentfc 22

24 or c) the use of a buffer contanng catonc speces wth a hgh affnty for the sorbent functonal groups. a b c Introducton of low concentratons of hgh surface affnty ons causes analyte eluton The caton-exchange nteracton s facltated by a solvent envronment that s smultaneously the converse of each of the condtons a. b. and c. above; that s, a solvent of low onc strength, at a ph such that both the analyte groups and the sorbent groups are charged, and f contanng other catons, these catons have a lower affnty for the sorbent than the analyte functonal group catons. Most pharmaceutcal bologcal samples are aqueous n nature. A protc envronment s a necessty for on-exchange to occur, and water s hghly protc, offerng a good startng pont for caton-exchange SPE. However, some bologcal samples (especally urne and certan cell-growth buffers) exhbt very hgh salt (very hgh onc strength) and at the very least requre sgnfcant dluton to reduce the onc strength pror to applcaton to an SPE column. The most common sorbents used n caton-exchange SPE have ether carboxylc acd or sulfonc acd groups on the surface. These groups can be onzed va a sutable ph adjustment, thus allowng postvely charged catonc analytes to retan. These sorbents are typcally avalable n both slca-based and polymer-based chemstres. Typcal strong caton exchange sorbents such as sulfonc acd phases are onsed over the whole ph range. Weaker sorbents such as the carboxylc acd derved phases can have ther degree of onzaton (and hence retenton strength) altered over a wde ph by manpulatng the soluton ph. Crawford Scentfc 23

25 At hgh ph values, all sorbent groups are nononsed At medum ph values, some sorbent groups are onsed At hgh ph values, all sorbent groups are onsed All slca-based sorbents, ncludng unbonded slca tself, also are capable of beng employed as weak caton-exchange surfaces, snce resdual slanol groups present on the surface are weakly acdc. If bonded slcas are to be used, extractons typcally work best wth sorbent havng short bonded groups (such as propylcyano), snce these shorter chans provde more access to the slanol groups as compared to long chans (such as C18) whch stercally block most of the resdual slanols. At hgh ph values, all sorbent groups are nonnon-onsed At medum ph values, some sorbent groups are onsed At hgh ph values, all sorbent groups are onsed At hgh ph values, all sorbent groups are onsed Acdc alumna may also be used qute effectvely as a caton-exchange sorbent. However, the user should pay careful attenton to the qualty of the alumna snce these materals often vary wdely n propertes batch- to-batch. All slca-based sorbents, ncludng unbonded slca tself, also are capable of beng employed as weak caton-exchange surfaces, snce resdual slanol groups present on the surface are weakly acdc. If bonded slcas are to be used, extractons typcally work best wth sorbent havng short bonded groups (such as propylcyano), snce these shorter chans provde more access to the slanol groups as compared to long chans (such as C18) whch stercally block most of the resdual slanols. Crawford Scentfc 24

26 Sample Pretreatment In addton to sample fltraton, f requred, and dsrupton of proten bndng, t s necessary pror to caton-exchange extracton to prepare a sample such that t exhbts the followng two prmary qualtes: a) the lowest onc strength possble,and b) a ph such that the analytes n the sample exst n a charged state. If the sample s serum or plasma, whch have relatvely low onc strengths to start, smple dluton of the sample wth water or a very low onc strength buffer may suffce to reduce the orgnal sample s onc strength suffcently for retenton. In some cases, such as wth urne samples, the onc strength s so hgh that t may be necessary to use a non-polar SPE procedure to remove excess ons pror to the caton-exchange procedure. Ths s often a very smple approach, snce norganc ons are readly washed off of non-polar phases wth smple buffers, n most cases leavng the analytes retaned. After analyte eluton from the non-polar sorbent, the low-onc strength extract may be dluted wth water or a buffer, and appled to the caton-exchange sorbent, where the analytes are agan retaned and reconcentrated. No retenton analyte n uncharged form Approprate ph adjustment of the sample to charge the onc groups on the sample s necessary to get qualty retenton on the caton-exchange sorbent. Basc or anonc groups on the analytes, whch are the typcal targets for a caton-exchange extracton, are onzed by droppng the sample ph to a mnmum of two ph unts below the pka of the relevant group. It s recommended to perform ths ph adjustment wth the strongest acd or the most powerful buffer possble so as to mnmze the ncrease n onc strength of the sample. For example, t s lkely to take a much smaller quantty of hydrochlorc acd to drop the ph of a typcal amne than of a weaker acd such as acetc acd. Crawford Scentfc 25

27 Good retenton analyte n charged form Sorbent Condtonng, Equlbraton and Sample Loadng Sorbent condtonng for caton-exchange SPE s smlar to that used for non-polar SPE. In most cases, the sorbent bed s frst treated wth a few bed volumes of a water-mscble organc solvent, such as methanol, acetontrle, or THF. Ths s typcally followed by a few bed volumes of pure water, to prepare the sorbent for the equlbraton step. Sorbent actvated (b) usng a non-polar organc solvent Sorbent equlbraton n caton-exchange SPE must accomplsh two man purposes: a) to convert the sorbent counter-on to one that s easly dsplaced by the analytes, and b) to adjust the sorbent ph to an approprate value so that the sorbent groups are charged. Crawford Scentfc 26

28 At hgh ph values, all sorbent groups are nononsed At medum ph values, some sorbent groups are onsed At hgh ph values, all sorbent groups are onsed To facltate analyte retenton on the caton-exchange sorbent, t s necessary to equlbrate wth a buffer or a salt soluton contanng a caton of lower affnty for the sorbent than the analyte group. Ths counter-on wll be most readly dsplaced by the analyte caton group. The lowest affnty caton avalable s hydrogen on (H+), and many caton-exchange sorbents may be purchased n the hydrogen form. If the natve counteron s not hydrogen and must be converted, ths s best acheved by washng wth an acd, such as hydrochlorc, acetc, or formc. However, care should be taken n the use of very strong acds such as hydrochlorc, snce these acds may degrade the ntegrty of a slcabased sorbent. a b Counter on too retentve complete analyte breakthrough For ph adjustment of an acdc sorbent, t s necessary to rase the ph of the sorbent to a mnmum of two ph unts above the pka of the sorbent n order to onze the sorbent groups. The pka values for most commercal sorbents are avalable from the respectve sorbent manufacturer. Crawford Scentfc 27

29 Sutable counter on complete analyte retenton Caton-exchange s extremely senstve to sample loadng speed. In partcular, t s very easy to pass the sample through the sorbent bed too quckly, n whch case breakthrough (nadequate analyte retenton) s lkely. A common reference pont for sample applcaton to a caton-exchange sorbent s no faster than 1 mlllter per mnute for a 100 mllgram sorbent bed. Ths speed lmtaton s a consequence of the pont-to-pont nature of onexchange,.e., a very specfc ndvdual group on an analyte molecule must encounter a very specfc group on the sorbent n order for retenton to occur. Washng and Analyte Eluton The caton-exchange retenton mechansm s facltated by a) low-onc strength buffers, b) buffers at a ph such that both the analyte and the sorbent reman charged, and c) organc solvents, whch have lttle or no effect on the onc retenton mechansm. Any of these reagents make excellent wash solvents for caton-exchange. Conversely, analyte eluton s facltated by a) hgh-onc strength buffers, b) buffers at a ph such that ether the analyte or the sorbent are neutralzed, or c) buffers contanng a counter-on that has a very hgh affnty for the sorbent (much hgher than the analyte caton group affnty for the sorbent). When usng hgh-onc strength as the eluton mechansm, t s often mportant to consder compatblty of the eluton buffer wth the fnal analytcal technque to be used. LC/MS systems, for example, do not cope well wth hgh onc strength samples, so t may be necessary to dry down and reconsttute the eluton solvent. In ths case, a volatle buffer salt, such as ammonum acetate should be used rather than a non-volatle salt such as sodum chlorde. Crawford Scentfc 28

30 Analyte eluton facltated by hgh onc strength buffer ph adjustment to facltate analyte eluton offers two possbltes: a) neutralzaton of the analyte groups, by rasng the ph to a mnmum of two ph unts above the analyte pka, or b) neutralzaton of the sorbent bed, by lowerng the ph to a mnmum of two ph unts below the sorbent pka. If possble, neutralzaton of the analyte groups s desred versus neutralzaton of the sorbent, snce sorbent neutralzaton wll release all speces retaned on the sorbent durng the extracton protocol, whch may nclude undesred contamnants. a b Analyte eluton facltated through analyte neutralsaton Crawford Scentfc 29

31 a b Analyte eluton facltated by neutralsaton of the sorbent bed The thrd approach to analyte eluton s to use a buffer contang a counter-on that has an extremely hgh affnty for the sorbent surface groups. These hgh-affnty catons can readly dsplace the analyte caton groups, whch may have much lower affnty for the sorbent, from the surface. Examples of these catons are lsted n the reference table of counter-ons at the end of the course and n the course manual. Analyte eluton facltated by counter on wth hgh affnty for the sorbent Anon-Exchange SPE Overvew Anon-exchange SPE s the extracton process where molecules wth acdc (anonc) functonal groups are extracted from predomnantly aqueous matrces onto sorbents wth basc (catonc) surface groups. Extracton of acdc molecules Crawford Scentfc 30

32 In anon-exchange SPE, the retenton mechansm s the nteracton of charged, anonc groups on the analytes of nterest and charged, catonc functonal groups on the sorbent, va electrostatc (onc) nteractons. These nteractons are dsrupted (causng eluton of the analytes) by one of three prmary methods: a) hgh onc strength, n whch the presence of large numbers of ons n the eluton buffer creates competton wth the analyte groups for the sorbent groups Analyte eluton facltated by hgh onc strength buffer Crawford Scentfc 31

33 b) a ph change, n whch ether the charged analyte groups are neutralzed, or the charged sorbent groups are neutralzed Analyte eluton facltated by neutralsaton of the sorbent bed or c) the use of a buffer contanng anonc speces wth a hgh affnty for the sorbent functonal groups. Analyte eluton facltated by hgh sorbent affnty counter ons Crawford Scentfc 32

34 The anon-exchange nteracton s facltated by a solvent envronment that s smultaneously the converse of each of the condtons a. b. and c. above; that s, a solvent of low onc strength, at a ph such that both the analyte groups and the sorbent groups are charged, and f contanng other anons, these anons have a lower affnty for the sorbent than the analyte functonal group anons. Most pharmaceutcal bologcal samples are aqueous n nature. A protc envronment s a necessty for on-exchange to occur, and water s hghly protc, offerng a good startng pont for anon-exchange SPE. However, some bologcal samples (especally urne and certan cell-growth buffers) exhbt very hgh salt (very hgh onc strength) and at the very least requre sgnfcant dluton to reduce the onc strength pror to applcaton to an SPE column. The most common sorbents used n anon-exchange SPE have some type of amne group on the surface; prmary, secondary, tertary, quaternary and multple amne surfaces are all avalable. These groups can be onzed va a sutable ph adjustment, thus allowng negatvely charged anonc analytes to retan. These sorbents are typcally avalable n both slca-based and polymer-based chemstres. Low ph values, all basc functonal groups are onsed Hgh ph values, all basc functonal groups are nononsed Basc alumna may also be used qute effectvely as a caton-exchange sorbent. However, the user should pay careful attenton to the qualty of the alumna snce these materals often vary wdely n propertes batch-to-batch. Sample Pretreatment In addton to sample fltraton, f requred, and dsrupton of proten bndng, t s necessary pror to anon-exchange extracton to prepare a sample such that t exhbts the followng two prmary qualtes: a) the lowest onc strength possble,and b) a ph such that the analytes n the sample exst n a charged state. If the sample s serum or plasma, whch have relatvely low onc strengths to start, smple dluton of the sample wth water or a very low onc strength buffer may suffce to reduce the orgnal sample s onc strength suffcently for retenton. In some cases, such as wth urne samples, the onc strength s so hgh that t may be necessary to use a non-polar SPE procedure to remove excess ons pror to the caton-exchange procedure. Ths s often a very smple approach, snce norganc ons are readly washed off of non-polar phases wth smple buffers, n most cases leavng the analytes retaned. After analyte eluton from the non-polar sorbent, the low-onc strength extract may be dluted wth water Crawford Scentfc 33

35 or a buffer, and appled to the anon-exchange sorbent, where the analytes are agan retaned and reconcentrated. Approprate ph adjustment of the sample to charge the onc groups on the sample s necessary to get qualty retenton on the anon-exchange sorbent. Acdc or catonc groups on the analytes, whch are the typcal targets for an anon-exchange extracton, are onzed by rasng the sample ph to a mnmum of two ph unts above the pka of the relevant group. It s recommended to perform ths ph adjustment wth the strongest base or the most powerful buffer possble so as to mnmze the ncrease n onc strength of the sample. For example, t s lkely to take a much smaller quantty of ammonum hydroxde to rase the ph of a typcal acd than of a weaker base such as trethyl amne. Poor analyte retenton as soluton ph dctates analyte s unchanged Maxmum analyte retenton as soluton ph dctates both analyte and sorbent are charged Sorbent Condtonng, Equlbraton and Sample Loadng Sorbent condtonng for anon-exchange SPE s smlar to that used for non-polar SPE. In most cases, the sorbent bed s frst treated wth a few bed volumes of a water-mscble organc solvent, such as methanol, acetontrle, or THF. Ths s typcally followed by a few bed volumes of pure water, to prepare the sorbent for the equlbraton step. Crawford Scentfc 34

36 Sorbent equlbraton n anon-exchange SPE must accomplsh two man purposes: a) to convert the sorbent counter-on to one that s easly dsplaced by the analytes, and b) to adjust the sorbent ph to an approprate value so that the sorbent groups are charged. To facltate analyte retenton on the anon-exchange sorbent, t s necessary to equlbrate wth a buffer or a salt soluton contanng an anon of lower affnty for the sorbent than the analyte group. Ths counter-on wll be most readly dsplaced by the analyte anon group. The lowest affnty anons avalable are fluorde and hydroxde ons. Most anon-exchange sorbent are not avalable n these forms, so the counter-ons must be converted. Ths s best acheved by washng wth a sutable salt at hgh onc strength; for example, f converson to fluorde s desred the sorbent may be equlbrated wth hgh molarty sodum fluorde. For ph adjustment of a basc sorbent, t s necessary to lower the ph of the sorbent to a mnmum of two ph unts below the pka of the sorbent n order to onze the sorbent groups. The pka values for most commercal sorbents are avalable from the respectve sorbent manufacturer. Anon-exchange s extremely senstve to sample loadng speed. In partcular, t s very easy to pass the sample through the sorbent bed too quckly, n whch case breakthrough (nadequate analyte retenton) s lkely. A common reference pont for sample applcaton to a caton-exchange sorbent s no faster than 1 mlllter per mnute for a 100 mllgram sorbent bed. Ths speed lmtaton s a consequence of the pont-to-pont nature of onexchange,.e., a very specfc ndvdual group on an analyte molecule must encounter a very specfc group on the sorbent n order for retenton to occur. Washng and Analyte Eluton The anon-exchange retenton mechansm s facltated by a) low-onc strength buffers, b) buffers at a ph such that both the analyte and the sorbent reman charged, and c) organc solvents, whch have lttle or no effect on the onc retenton mechansm. Any of these reagents make excellent wash solvents for anon-exchange. Low onc strength buffer no analyte eluton Crawford Scentfc 35

37 Conversely, analyte eluton s facltated by a) hgh-onc strength buffers, b) buffers at a ph such that ether the analyte or the sorbent are neutralzed, or c) buffers contanng a counter-on that has a very hgh affnty for the sorbent (much hgher than the analyte anon group affnty for the sorbent). When usng hgh-onc strength as the eluton mechansm, t s often mportant to consder compatblty of the eluton buffer wth the fnal analytcal technque to be used. LC/MS systems, for example, do not cope well wth hgh onc strength samples, so t may be necessary to dry down and reconsttute the eluton solvent. In ths case, a volatle buffer salt, such as ammonum acetate should be used rather than a non-volatle salt such as sodum chlorde. a b Analyte eluton facltated by hgh onc strength buffer ph adjustment to facltate analyte eluton offers two possbltes: a) neutralzaton of the analyte groups, by lowerng the ph to a mnmum of two ph unts below the analyte pka, or b) neutralzaton of the sorbent bed, by rasng the ph to a mnmum of two ph unts above the sorbent pka. If possble, neutralzaton of the analyte groups s desred versus neutralzaton of the sorbent, snce sorbent neutralzaton wll release all speces retaned on the sorbent durng the extracton protocol, whch may nclude undesred contamnants. Analyte eluton through analyte neutralsaton Crawford Scentfc 36

38 Analyte eluton through surface neutralsaton The thrd approach to analyte eluton s to use a buffer contanng a counter-on that has an extremely hgh affnty for the sorbent surface groups. These hgh-affnty anons can readly dsplace the analyte anon groups, whch may have much lower affnty for the sorbent, from the surface. Ths effect s dramatcally more pronounced n anon-exchange than n caton-exchange, and therefore can be hghly useful. Examples of these anons are lsted n the reference table of counter-ons at the end of the course and n the course manual. Mnmum pka Dfferental Analyte eluton usng strong counter on The ssue of frst sample ph adjustment, then sorbent bed ph adjustment, brngs out a very mportant ssue when performng on-exchange SPE, whether caton or anon exchange that of mnmum pka dfferental. As dscussed earler n the course, for an onc group to be substantally n ether the charged or neutral form, the ph of the surroundng envronment must be a mnmum of two ph unts away from the pka of the Crawford Scentfc 37

39 group. For a caton to be charged, the ph must be two unts above the pka, and for an anon the ph must be two ph unts below the pka. Ths requrement may create dffcultes f not consdered carefully. For example, assume that t s desred to perform caton exchange on a molecule that has a pka of 6.7, usng an anonc (caton-exchange) sorbent wth a pka of 4.5. Accordng to our two-ph unt premse, for the analyte to be charged, we must be at ph 4.5 or below, but for the sorbent to be charged we must be at ph 6.5 or above. Clearly these two condtons cannot be met smultaneously. The corollary to ths s that to perform on-exchange properly, we need a) a four ph unt dfferental between the pka value of our anon and caton, and b) the caton must have a pka hgher than the anon. Ths stuaton may well dctate whch sorbents are chosen for the specfc analytes to be extracted, based on the known pka of the analyte and the pka of the sorbent. Crawford Scentfc 38

40 Crawford Scentfc 39

41 Effect of ph n analyte retenton Mxed-Mode SPE Overvew Mxed-mode SPE s an extracton approach nvolvng sorbents whch are desgned to exhbt two or more prmary nteractons for analyte retenton. The use of multple retenton mechansms offers unque advantages n terms of selectvty and breadth of applcablty to many dfferent chemcal structures. Crawford Scentfc 40

42 Commercal mxed-mode sorbents may be slca or polymer-based, and are typcally produced n one of two ways ether by bondng the sorbent concurrently wth two dfferent functonal group chemstres, or by blendng dscrete sorbent chemstres n approprate ratos to gve the combnaton of retentve propertes desred. Although the two approaches result n materals of smlar propertes, t s generally recognzed that the blendng approach s preferred for two reasons: a) t s easer to bond to a surface or synthesze a sngle functonal group chemstry reproducbly than to do so wth multple functonal groups, and b) f dfferent retentve propertes are requred, t s much more flexble to smply blend sngle functonal group sorbents n dfferent ratos than to create an entre new synthess to acheve the same effect wth multple functonal groups on a gven surface. Crawford Scentfc 41

43 The most common mxed-mode sorbents have a hydrophobc functonal group n combnaton wth an on-exchange functonal group. Hydrophobc groups may vary from short chan, less retentve thus more selectve - materals (such as a C2 surface) to hghly retentve surfaces, such as C18. Ion-exchangers may be ether caton or anon exchangers, or even both. Hydrophobc chemstres par well wth on-exchange chemstres, snce both allow effectve loadng of analytes n smlar matrces; for example, hghly aqueous samples. Ths s an deal condton for pharmaceutcal bologcal samples such as serum, plasma, blood, urne, CSF or varous buffers. The standard approach n employng a mxed-mode sorbent s to use the sorbent to extract an analyte that exhbts functonal group characterstcs capable of nteractng wth both of the actve modes n the sorbent. For example, a mxed mode sorbent contanng a caton exchanger and a hydrophobc chemstry works well wth a basc analyte that also has some hydrophobcty. Thus when the analyte s loaded, the caton exchanger nteracts wth the basc functonal group on the analyte, and the hydrophobc sorbent group nteracts wth the hydrophobc analyte groups. In fact, ths partcular combnaton (.e., hydrophobc and caton-exchange) s by far the most popular sorbent used n pharmaceutcal extracton of basc drugs. For the steps descrbed to follow, we wll assume ths s the chemstry combnaton wth whch we are dealng. In order to effectvely elute an analyte that s beng retaned on a mxed-mode surface va two separate mechansms, t s necessary to use an eluton solvent that smultaneously dsrupts both of the retenton mechansms. Ths concept s key to the strength of a mxedmode sorbent n provdng sample extracts of extraordnary cleanlness. Crawford Scentfc 42

44 Crawford Scentfc 43

45 Sample Pretreatment One of the advantages of mxed-mode SPE s that t s more forgvng than sngle-mode technques n terms of raw sample qualty. Multple retentve mechansms requre only that a sample s optmzed ntally to accommodate one of the actve sorbent mechansms. Wth a sngle mechansm only, the sample must be optmzed to accommodate that partcular mechansm. The problem wth ths may best be exemplfed by a hydrophobc, basc analyte from urne. Urne typcally carres a relatvely hgh onc strength. If ths sample s appled drectly to a caton-exchange sorbent, the analyte s lkely to experence breakthrough due to competng matrx ons. Sample dluton may help but may be nsuffcent to reduce the onc strength adequately. However, f the same sample s appled to a mxed-mode caton-exchanger/hydrophobc sorbent, the analyte may be retaned by the hydrophobc mechansm wthout regard to the onc character of the sample. Ths s analogous to applyng the sample to a sorbent wth hydrophobc propertes only, but the mxed-mode wll contrbute further benefts n subsequent steps. Crawford Scentfc 44

46 Regardng the specfc sample preparaton steps, as wth all samples, t may frst be necessary to address the ssue of proten bndng or fltraton removal of partculates. As far as alterng the chemstry of the sample, we must prepare the sample accordng to the mechansm we wsh to use. So, for example, f we are usng a combnaton onexchanger/hydrophobc mxed-mode sorbent, and we wsh to use the on-exchange mechansm, the sample must be prepared as f we were usng an on-exchange-only sorbent. Ths mght nvolve sample dluton and ph adjustment. Sorbent Condtonng Equlbraton and Sample Loadng Sorbent condtonng for mxed-mode SPE s a combnaton of that used for both hydrophobc sorbents and on-exchange sorbents. Typcally the surface s frst condtoned wth methanol, acetontrle or THF, to wet the sorbent. Sample condtonng Then the excess organc solvent s removed usng a sutable buffer. In ths case the buffer may also be of a ph to onze the on-exchange groups, but ths s only necessary durng equlbraton f the on-exchange mechansm s desred to be actve when the sample s Crawford Scentfc 45

47 frst appled. Also, f a counter-on change s desred for the on-exchanger, ths should be performed. Equlbraton buffer used to adjust soluton ph ensurng caton exchanger s fully onsed The requrements for the sample loadng step, as wth the prevous steps, s dependent on whch mechansm s to be employed as the prmary retentve mechansm. If on-exchange s to be used, the sample should be appled at a rate comparable to that used for onexchange alone, that s, relatvely slowly. If the prmary ntal retentve mechansm s the hydrophobc mechansm, then the sample may be loaded much faster. Washng and Analyte Eluton Washng and eluton are two steps n mxed mode whch exemplfy the strength of the technque n provdng very clean extracts. The prncple here s straghtforward snce our target analytes are retaned (presumably) by both retenton mechansms, we can selectvely wash off nterferences that are retaned only by a sngle mechansm. Therefore, n a mxed-mode extracton we typcally employ two separate wash steps. For example, agan assumng that our analyte s both basc and hydrophobc, and s retaned on a mxed mode sorbent contanng both caton-exchange and hydrophobc chemstres, we can frst use a wash step whch removes only matrx components that have onc character. An example of ths mght be salts from a urne matrx. Ths step mght use, for example, a hgh onc strength buffer. Such a wash step would have no nfluence on the retenton of the analytes va a hydrophobc mechansm. Then, as a second wash, we mght use a pure organc solvent, whch wll remove matrx components wth only hydrophobc character (no onc retenton), wthout affectng the onc retenton of the analytes. Usng ths double-wash approach very effectvely removes many nterferences from the sorbent wthout affectng the analytes. Crawford Scentfc 46

48 Crawford Scentfc 47

49 For effectve analyte eluton, we must employ a solvent that dsrupts both retenton mechansms smultaneously. For mxed-mode sorbents wth on-exchangers and hydrophobc groups, ths s typcally an organc solvent (such as methanol, acetontrle, or THF), modfed wth a strong acd or base to negate the onc retenton. So, for example, a typcal eluton solvent mght be methanol contanng a few percent of ammonum hydroxde or hydrochlorc acd. As wth other retenton mechansms, maxmum extract purty s acheved by a) washng wth the strongest solvent possble that does not elute the analytes, and b) elutng wth the weakest solvent possble that gves hgh analyte recovery, yet leaves behnd on the sorbent as many remanng nterferences as possble. Crawford Scentfc 48

50 Summary Mxed-mode extracton s often somewhat confusng to users, yet can be understood qute smply by recallng the followng gudelnes: Be certan to dentfy whch sorbent mechansms are represented by any gven commercal product. Although there are many dfferent combnatons of sorbent chemstres avalable, by far the most common are a hydrophobc chemstry pared wth ether an anon or a caton-exchange chemstry. Consder the retentve mechansms as ndvdual chemstres actng together, and treat the sorbent approprately. For example, condtonng steps to prepare the caton-exchange element of a mxed mode sorbent are the same as those prescrbed for a caton-exchange sngle mechansm sorbent. All of the ph, onc strength, and counter-on prncples apply equally. The hydrophobc element should also be treated n a smlar, analogous manner to a standalone hydrophobc sorbent. Bear n mnd that the two retentve mechansms may help to retan an analyte from a matrx envronment that would be ncompatble wth a sngle mechansm sorbent. Agan, the prncple here s to use the favoured mechansm to overcome matrx lmtatons untl the analyte s retaned and the uncooperatve nterferences removed through washng. Use aggressve wash solvents that only ntercede n one of the two retenton mechansms, so as to preserve analyte retenton va the other mechansm. For maxmum eluton effectveness, be sure to use a solvent that smultaneously dsrupts both retenton mechansms. Crawford Scentfc 49

51