Sample Preparation. Primary Sample Preparation Techniques

Transcription

1 Sample Preparaton Prmary Sample Preparaton Technques 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 present sample dluton as the most basc of all sample preparaton technques and present ts basc prncples To descrbe the basc prncples of centrfugaton and ultracentrfugaton To ntroduce proten precptaton and dscuss relevant aspects about t To lst and dscuss the most mportant extracton approaches to sample preparaton Objectves At the end of ths Secton you should be able to: Explan concepts of proten precptaton Choose approprate extracton approaches for varous analyte and sample matrx types Decde when to use centrfugaton (or ultracentrfugaton) and understand what could be acheve t Identfy stuatons n whch smple sample preparaton technques (dluton, fltraton, centrfugaton,..) are suffcent to overcome practcal problems

3 Content Prmary Sample Prep Technques n Pharmaceutcals 3 Sample Dluton 5 Sample Fltraton and Ultrafltraton 6 Centrfugaton and Ultracentrfugaton 8 Proten Precptaton 9 Extracton Approaches to Sample Preparaton 10 Selectvty 11 The Partton Coeffcent 13 Lqud / lqud Extracton 14 Support-Asssted Lqud/Lqud Extracton 15 Sold Phase Extracton 17 Crawford Scentfc 2

4 Prmary Sample Prep Technques n Pharmaceutcals In pre-clncal and clncal trals, most of the samples are fundamentally aqueous n nature. Snce dfferent sample preparaton technques have dfferng degrees of functonalty and effectveness due to the nature of the orgnal sample matrx, the aqueous character of the samples often reduces the number of potental sample preparaton technques lkely to be used. All common sample preparaton technques that reduce the number of sample components depend on dscrmnaton by a) molecular sze, b) solublty, or c) functonal group characterstcs, especally as related to chromatographc propertes. By far the most common sample preparaton technques used n modern pharmaceutcal boanalyss are proten precptaton, lqud/lqud extracton, and sold phase extracton. Wthn each of these technque categores, consderaton must be gven to the aqueous nature of the startng samples such that optmum effectveness s acheved wth any gven technque. For example, lqud/lqud extracton performed on aqueous samples s lmted to the use of water-mmscble extracton solvents. Smlarly, wth SPE, the most commonly employed extracton mechansms are those best suted to aqueous samples, n partcular non-polar or on-exchange. 1 2 Lowerng the sample ph or addng organc solvent can cause protens n soluton to precptate, leavng non-bound analytes and potental nterferents n soluton Protens Proten Precptaton Crawford Scentfc 3

5 Agtaton / mxng causes the analyte to partton nto the organc phase from where t can be decanted. Solvent polarty s mportant here as the am s to leave as many nterferents behnd n the aqueous layer as possble. Lqud-Lqud Extracton In ths smple Sold Phase Extracton (SPE) experment the sample passes through the cartrdge whlst the nterferng components are retaned on the sorbent achevng smple and effectve sample clean up. Careful choce of the sorbent chemstry s mportant Sold Phase Extracton Crawford Scentfc 4

6 Sample Dluton The most basc of all sample preparaton technques s smple dluton of the orgnal sample pror to analyss, usng an approprate dluton solvent. Ths technque has the rather severe lmtaton of not actually removng contamnatng speces from the sample, as wth other, more sophstcated technques. Sample dluton may, however, serve to accomplsh one of two mportant functons wthn sample preparaton. The frst s smply to reduce the vscosty of the sample, allowng the analytcal nstrument to handle the sample n a more facle manner. For example, many autosampler devces have a dffcult tme handlng hghly vscous samples reproducbly, and dluton may mprove ths. A second reason to dlute a sample s to change the basc character of the sample matrx to make the sample more chemcally compatble wth the analyss. For example, n gradent lqud chromatography, t s often undesrable to ntroduce a sample n a very strongly elutng solvent (hgh organc content) nto the ntal gradent condtons (usually a weakly elutng hghly aqueous solvent mxture). Ths phenomenon leads to poor chromatographc peak shape. Dluton to reduce the strength of the sample may correct ths problem qute easly. Dluton wth an approprate solvent can help to overcome facle processng dffcultes and mprove chromatographc peak shape. Sample Dluton Crawford Scentfc 5

7 Sample Fltraton and Ultrafltraton Lke sample dluton, sample fltraton s a very smple technque that mproves the qualty of a sample by removng partculate matter that may be ncompatble wth the analytcal technque. Unfltered partculates can often clog nstrument nterfaces and HPLC columns, creatng hgh backpressure or blockng devces altogether. At the very least, ths problem s lkely to reduce operatng lfetmes of columns and even the nstruments themselves. Unfortunately, sample fltraton does lttle to purfy the sample from a chemcal standpont by removng undesred contamnant speces from soluton. Therefore, n most case fltraton alone s nsuffcent to provde adequate sample qualty for a typcal boanalyss. An excepton to ths s known as ultrafltraton. Ultrafltraton uses membrane flters of very controlled, low specfc porosty. These membranes are capable of removng large speces exstng n soluton from the soluton, most notably bomolecules such as protens. Ultrafltraton s a legtmate purfcaton technque n ts own rght the prmary lmtatons beng the facle processng of samples, and the ablty to automate the process. Crawford Scentfc 6

8 Sample partculate materal can block HPLC columns leadng to hgh system back pressure and shortened column lfetme. Fltraton Process Crawford Scentfc 7

9 Centrfugaton and Ultracentrfugaton Centrfugaton s a technque that allows for removal of partculate speces from samples pror to analyss. Because centrfugaton alone does not perform a chemcal purfcaton of a sample, t s typcally nsuffcent alone as a preparaton technque. However, centrfugaton s a common adjunct to other technques, ncludng proten precptaton, where centrfugaton allows for facle separaton of the precptated protens from the sample. As wth fltraton, there s a varant of centrfugaton that s more effectve as a sample preparaton technque ultracentrfugaton. Ths nvolves centrfugaton of samples at speeds suffcently hgh that very large molecules n soluton (for example, protens) may actually be removed from the sample by centrfugal force. However, the nherent beneft of ths approach s offset by the expense of the centrfugal devces requred to mplement the process. Crawford Scentfc 8

10 Proten Precptaton Proten precptaton s a commonly-employed technque for relatvely crude but rapd sample clean-up. The prncple of proten precptaton s qute smple, n that many protens are soluble n a sample only under a very lmted range of solvent envronments. If the solvent envronment s altered n an approprate manner, the protens wll come out of soluton (sometmes referred to as a proten crash ), and can then be removed from the sample by fltraton or centrfugaton. Proten precptaton can be accomplshed by a number of methods, ncludng addton of an organc solvent to the sample, addton of a non-onc surfactant, addton of norganc salts, or addton of certan metals. By far the most common of these s addton of an organc solvent, usually acetontrle n a quantty to compose a mnmum of 30% of the sample solvent consttuton. Perchlorc acd and trchloroacetc acd are also sometmes used. Crawford Scentfc 9

11 Although proten precptaton can be very effectve n proten removal, t does not address the presence of other sample contamnants that may cause analytcal problems, such as lpds. Proten Precptaton Extracton Approaches to Sample Preparaton Extracton approaches to sample preparaton nvolve transfer of ether target analytes or undesred sample contamnants from one chemcal phase to another. Of necessty, a mnmum of two phases must be used to acheve a separaton. A chemcal phase may be a lqud, a sold, or a gas, although most sample preparaton methods used n pharmaceutcal boanalyss employ ether two lquds or a lqud and a sold. If ths transfer s accomplshed on a dfferental bass, a purfcaton of one speces compared to the other s acheved. In some cases, very complex samples contanng many dfferent components may be reduced to hghly purfed extracts. The more effectve a procedure s at separatng the desred speces from the undesred contamnants, the more selectve t s sad to be. Crawford Scentfc 10

12 Non Selectve Extracton Selectve Extracton Hghly Selectve Extracton Selectvty Selectvty refers to the ablty of a sorbent or an extracton protocol to separate or dscrmnate between the compound or compounds of nterest and other components of the orgnal sample to be purfed. When we state that a sorbent or protocol s hghly selectve, ths means the extracton wll remove a large proporton of the contamnants from the sample. The result of ths wll be a hghly purfed, hgh qualty sample. Crawford Scentfc 11

13 Conversely, when we state that a sorbent or protocol has low selectvty, ths means that the extracton wll remove only a mnmal amount of the contamnants from the sample. The result of ths wll be a less purfed, poorer qualty sample. There are advantages and dsadvantages of both hgh and low selectvty technques. In general, a low selectvty technque may be applcable to a very large range of compounds. Low selectvty technques such as ths are often referred to as generc methods. A so-called generc method wll depend on a chemcal property that the analyte molecule may share n common wth many other compounds n the sample. Hgh selectvty technques are often applcable to only a lmted set of compounds. Due to ths, creaton of a hgh selectvty protocol may requre more method development tme than for a low selectvty protocol. Hgh selectvty protocols rely on analyte chemstry that s shared by very few other components of the sample. Another mportant concept n extracton technques s the partton coeffcent. Selectve/Non Selectve Extracton Crawford Scentfc 12

14 The Partton Coeffcent When usng extracton approaches to sample preparaton, a useful parameter to understand s the degree to whch any gven speces prefers to be n one of the chemcal phases versus the other. Ths s known as the partton coeffcent, and s also referred to as the equlbrum constant. If we consder the orgnal phase (usually the orgnal sample) to be phase 1, and the extractng phase to be phase 2, the partton coeffcent for a speces X s defned to be the concentraton of X n phase 2 dvded by the concentraton n phase 1, once the system has reached an extracton equlbrum. Ths dstrbuton between two phases s, of course, nfluenced by the specfc chemcal nature of the phases, nteractng wth the propertes of the speces of nterest. Crawford Scentfc 13

15 In order to acheve hgh recoveres of extracted analytes n pure extracts, t s mportant for partton coeffcents to be 1) as hgh as possble, ensurng essentally complete resdence n ether one phase or another, and 2) to be as dfferent as possble for the speces to be separated. Indeed, t may be sad that the goal of a sklled extracton chemst s to become as adept as possble at manpulatng partton coeffcents of the varous speces wthn a sample. We are now ready to ntroduce the two most mportant and common extracton approaches n sample preparaton - lqud/lqud extracton, and sold-phase extracton ( SPE ). Lqud / lqud Extracton Lqud/lqud extracton s a long-standng approach to sample preparaton, not only n boanalytcal pharmaceutcal work, but also n organc synthess. Lqud/lqud extracton s based on dfferental solublty of varous sample components. In prncple, lqud/lqud extracton s executed by addng a solvent that s mmscble wth the orgnal sample solvent to the sample, creatng two separate layers. Ths system s Crawford Scentfc 14

16 then agtated by shakng or vortexng, and then allowed to separate agan nto the two startng layers. If, for example, the target analytes from the sample exhbt hgher solublty n the extractng solvent than n the orgnal sample solvent, they wll pass nto the extractng solvent, often dfferentally as compared to other orgnal sample components. Ths results n the extractng solvent layer contanng the target analytes n a more hghly purfed form than n the orgnal sample. Lqud/lqud extracton may also be used n the format where the sample contamnants, nstead of the target analytes, are extracted nto the added solvent. Ths also provdes a more purfed alquot of the target extracts, but n ths case the analytes reman n the orgnal startng sample solvent. Support-Asssted Lqud/Lqud Extracton An nterestng and useful varaton of lqud/lqud extracton s support-asssted lqud/lqud extracton. Compounds are separated on the same bass of dfferental solublty, as wth conventonal lqud/lqud extracton. However, nstead of smply usng two mmscble lquds n a contaner, n support-asssted lqud/lqud extracton, one lqud s dstrbuted over the surface of a sold support contaned n a chromatographc column format. The second, extractng solvent s then passed through the column, and compounds partton from the mmoblzed layer on the support nto the extractng solvent as t passes through the column. As n conventonal lqud/lqud extracton, ths approach may be used n one of two ways the target analytes may transfer nto the extractng solvent, leavng the contamnants behnd n the orgnal sample on the support, or contamnants may transfer nto the extractng solvent, leavng the target analytes behnd on the support. In the latter case, t s then necessary to recover the analytes by dsplacng the orgnal sample solvent from the support usng an approprate secondary solvent. Crawford Scentfc 15

17 Crawford Scentfc 16

18 Sold Phase Extracton Sold phase extracton, or SPE, s perhaps the most powerful sample preparaton technque n common use today. Among SPE s strengths are selectvty, flexblty, and hgh automaton potental. SPE nvolves the use of a chromatographc sorbent n a column format. A sample s passed through the column bed, analytes retan on the sorbent whle the sample matrx lqud passes through, then the sorbent bed s washed to remove undesred nterferences, and the purfed analytes subsequently eluted from the column. SPE may also be used to retan nterferences, allowng analytes to pass unretaned through the sorbent bed. SPE products are avalable n a varety of formats to accommodate dfferent sample szes and applcatons. The orgnal format used most commonly n pharmaceutcal sample preparaton s a small syrnge barrel, contanng 100 mllgrams or less of sorbent materal. Ths has more recently been somewhat dsplaced by a 96-well plate format, well suted for automaton and supported by many commercal automaton platforms. Crawford Scentfc 17

19 SPE Formats Crawford Scentfc 18

20 SPE Formats Crawford Scentfc 19

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