OD-H,OD-RH,AGP,ChiralpakIB,IA 柱使用指南

1、ChromTech“your chromatography specialists”Users GuideSeparation of chiral compounds onChiral-AGP Chiral-CBH Chiral-HSAqqSecond Edition800-822-5242 (LCGC) In MN (952) 431-6000 FAX (952) 431-6345Visit our Website: email: salesContentsThe CHIRAL-AGP column.3The CHIRAL-CBH column.3The CHIRAL-HSA column.

2、3Column Selection Guide.3Method Development.4Substance Index.7Applications.11Reference list CHIRAL-AGP.28Reference list CHIRAL-CBH.34Reference list CHIRAL-HSA.35Chiral Column Price List.35ates pure organic solvents, high temperatures and high and low pH.1-acid glycoprotein (AGP) is a very stable pro

3、tein, which toler-AGP is the chiral selector in the CHIRAL-AGP column. The selec-tor has been immobilized on spherical 5 is used in the reversed-phase mode. The CHIRAL-AGP column canm particles. The columnbe used for the resolution of an extremely broad range of chiralcompounds, such as amines (prim

4、ary, secondary, tertiary and qua-ternary ammonium), acids, esters, sulphoxides, amides, alcoholsetc. The very broad applicability is demonstrated in the applicationsection below and in the list of publications in the last part of theguide. In the applications you can find chromatograms together with

5、the chromatographic conditions.The enantioselectivity and the retention can easily be regulated bythe pH of the mobile phase, the buffer concentration and the natureand the concentration of the organic modifier.Stability of the CHIRAL-AGP columnThe stability of the AGP column has been tested using b

6、umadizon,an acidic drug, as test compound. In total (10% isopropanol in phosph. buffer pH 6.0) was pumped through30.5 liters of mobile phasethe column. jected.During the test 2030 samples of bumadizon were in-gram and the other one is the last chromatogram obtained in theOne of the chromatograms bel

7、ow is the starting chromato-test. No significant changes were observed.Cellobiohydrolase(CBH) is the chiral selector in the CHIRAL-CBHcolumn. CBH is a very stable enzyme, which has been immobi-lized onto spherical 5 reversed-phase mode. The column is preferably used for the sepa-m silica particles.

8、The column is used in theration of enantiomers of basic drugs from many compound classes.The retention and the enantioselectivity can be regulated by changesin pH, buffer concentration and the nature and the concentration oforganic modifier.The chiral selector used for this stationary phase is the h

9、uman se-rum albumin (HSA). The protein has been immobilized onto spheri-cal 5 mode. Enantiomers of preferentially acidic compounds can be re-m silica particles. The column is used in the reversed-phasesolved on the column. As for the other two columns retention andenantioselectivity can be regulated

10、 by changing the mobile phasecomposition, see above.Quality control of the columnsThe silica used for the manufacturing of the chiral columns is testedaccording to an extensive test protocol. When approved the silicasurface is modified. All the chemicals used for the surface modifi-cation are either

11、 purchased against certificate or tested and approvedby ChromTech. After surface modification a batch test is performed.If the test parameters are within the specifications, the batch is ap-proved and released for production of columns. The next step is thecontrol of the final product. Each column i

12、s tested to control sepa-ration efficiency, retention and resolution.CHIRAL-AGPExtremely broad applicability. Mostlikely the column with the broadestapplicability of all chiral columnsavailable.Separates all types of compounds:- amines (primary, secondary,tertiary and quaternary nitrogen)- acids (st

13、rong and weak)- non-protolytes (amides, esters,alcohols, sulphoxides, etc.)CHIRAL-CBHMore narrow applicability thanCHIRAL-AGP. Separates preferablycompounds containing nitrogenshydrogen accepting or hydrogen together with one or moreone or moredonating groups (alcohol, phenol,carbonyl, amide, ether,

14、 ester etc.).CHIRAL-HSAMore narrow applicability thanCHIRAL-AGP. Separates preferablyweak and strong acids, zwitterionicand non-protolytic compounds.As can be seen the columns overlap for some types of compounds;basic CHIRAL-CBH, acidic and non-protolytes can be separated on bothcompounds can be sep

15、arated on both CHIRAL-AGP andCHIRAL-AGP and CHIRAL-HSA. However, as CHIRAL-AGP isa column with an extremely broad applicability, this column shouldbe the first choice, if the analyte has not been resolved on any of thecolumns. There are, however, some types of componds where oneof the other columns

16、might be the first choice:CHIRAL-HSACHIRAL-CBH: very hydrophilic acids: very hydrophilic aminesSee p. 35 for a list of available column dimensions.The columns described here are reversed-phase columns giving manypossibilities to affect both the retention and the enantioselectivity.The (charged solut

17、es), hydrophobic interaction and hydrogen bonding.solutes are retained by three types of forces; ionic bindingThe relative contribution of the different forces to the retention ofthe solutes, depend of the nature of the analyte. Analytes containingcharged groups, hydrogen bonding groups and hydropho

18、bic partscan chiral selector. From this follows that a separation can be affectedbe retained by interaction with corresponding groups on theby:- pH- buffer concentration- type of buffer- organic modifier concentration- type of organic modifierMethod development schemesAll columns are delivered with

19、a method development scheme thatmakes the method development very simple. In this scheme youwill find the starting mobile phase to use for a certain type of com-pound. When you have the first result with the starting mobile phaseyou can simply follow the scheme which in most cases gives a base-line

20、separation.CHIRAL-AGPThe most important tool in method development is the pH. The rea-son is that by changing the pH the net charge of the chiral selectoras well as the charge of the solute can be changed, which affects theway the analyte interacts with the chiral selector. AGP has a lowisoelectric

21、point of 2.7. This means that using the column at pH 2.7gives a net charge of zero of the chiral stationary phase. Increasingthe pH from 2.7 up to 7 means that the degree of net negative chargeof the chiral selector increases. This gives the prerequisites for ionicbinding of positively charged solut

22、es, resulting in a high affinityand high retention of the solute. Reducing the pH towards the iso-electric point reduces the negative charge of the stationary phase,resulting in lower retention of the solute. A change of the net chargeof the chiral selector strongly affects the interaction between t

23、hesolute and the chiral stationary phase. It has been demonstrated thationic binding of amines to the AGP column is a very important typeof interaction for retention of this category of compounds. The sol-utes are also retained by hydrophobic interaction and hydrogen bond-ing. The relative influence

24、 of the different types of binding forcesdepends of the nature of the solute, i.e. what kind of structure ele-ments are present in the analyte.Below you will find examples of the effect of changing the compo-sition of the mobile phase, i.e. the pH, the modifier concentrationand the modifier nature e

25、tc.4Changing the pHWhen chromatographing hydrophobic amines a pH of 4-5 is pre-ferred compared to a pH of 7. The explanation to this finding is thatchromatography of the amine at a pH of 7, where the protein has astrong degree of net negative charge and the analyte is positivelycharged, gives a stro

26、ng ionic binding of the analyte. However, re-ducing the pH to the range 4-5 reduces the degree of net negativecharge of the protein (the analyte is still fully ionized) which givesa reduction of the ionic bonding of the analyte and the retention isstrongly reduced. For some compounds even a decrease

27、 to pH 6might give large improvements compared to pH 7.The chromatographed at pH 4 and 7. Note the very strong reduction ofpH effects are demonstrated below for propranolol,the retention and the improvement of the chromatographic perfor-mance at pH 4. See also the numerous application examples of co

28、m-pounds chromatographed at pH 4-5.The pH can also be an effective tool for affecting the resolution ofacids which is demonstreted below for The compound has been chromatographed at three different pH, 5,2-phenoxypropionic acid.6 and 7. The analyte is totally ionized (negatively charged) at pH 7,but

29、 the charge is reduced at lower pH since the pKa-value is about4. Furthermore, a decrease in pH reduces the degree of net negativecharge of the protein, resulting in higher retention due to reductionof the repulsion between the analyte and the chiral stationary phase.The bonding.solute is retained b

30、y hydrophobic interaction and hydrogenChanging the buffer concentrationBy changing the buffer concentration, it is possible to affect boththe retention and the enantioselectivity. Such effects have been ob-served for acids and for certain amines. The chromatograms belowan example for the acidic drug

31、 naproxen.Changing the modifier concentration2-propanol, most frequently used organic modifiers. Higher modifier concen-acetonitrile, methanol, ethanol and 1-propanol is thetration amines reducesenantioselectivity can be strongly improved by increasing the modi-and acids. the retention However, and

32、for the certain enantioselectivity types of acids for boththefier concentration, as is demonstrated for warfarinbelow.Conc. 2-propanol (%)k18104.731.33122.451.191.42140.761.531.57Changing the nature of the modifierBy changing from one organic modifier to another with differenthydrogen bonding proper

33、ties, i.e. from acetonitrile (hydrogen ac-cepting properties) to 2-propanol (hydrogen accepting and donat-ing properties), it is possible to strongly affect the enantioselectivityas demonstrated below for chiral selectivity, while acetonitrile gives a complete base-line reso-pindolol. Using 1-propan

34、ol results in nolution.CHIRAL-CBHThe majority of the compounds chromatographed on the CHIRAL-CBH column are amines. See the applications. The CBH column isused in the reversed-phase mode.The same type of mobile phases can be used on both the AGP andthe CBH columns. The retention and the enantioselec

35、tivity is af-fected by the pH, the buffer concentration, the nature and the con-centration of the organic modifier. The same types of forces areinvolved in the retention process of the solute as was described forthe AGP column above.Changing the pHA decrease in pH will result in decreasing retention

36、 and in mostcases lower enantioselectivity, as is demonstrated for epanolol be-low.Changing the modifier concentrationThe most widely used organic modifiers on the CBH column are 2-propanol and acetonitrile. Normally, increasing modifier concen-tration enantioselectivity. These effects are illustrat

37、ed below for results in reduction of the retention and increasingatenololand talinolol.Addition of an organic modifier has in almost all cases a positiveinfluence on the chromatographic performance compared to chro-matography in pure buffers. See below for laudanosine.pHk1k25.06.01.447.01.301.821.87

38、1.260.753.721.444.97Changing the modifier concentration2-propanol, 1-propanol and acetonitrile are frequently used modifi-ers on the CHIRAL-HSA column. A higher organic modifier con-centration reduces the retention. Normally, also the enantioselectivitywill decrease.These effects are exemplified bel

39、ow for kynurenine.However, for certain acidic compounds it has been observed thatthe enantioselectivity is increasing when an organic modifier is addedto the mobile phase as is demonstrated below for abscisic acid.Abscisic acid, effect of 2-propanolMobile phase: 100 mM sod. ph. b. pH 7.0% 2-propanol

40、k1k2013.621.964.563.371.261.92Abscisic acidAcebutolol-alanin-N-2-(3,4-dihydro-2H-1-benzo-pyran-3-yl)-ethyl methyl ester hydro-chlorideAlfuzosinAlimemazineAlprenololCHIRAL-AGPCHIRAL-CBHCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-AGP11111111111176, 129, 14912917, 304, 29, 1016, 12, 13, 29, 76, 101, 112Aminog

41、lutethimideAmlodipineAtenololAtropine8-Azaspiro4,5decane-7,9-dione-8-(2-(2,3- dihydro-1,4-benzodioxin-2-yl)-methylaminoethyl) monomethanesulfonateBendroflumethazideBenflourexBenzoinN-benzoyl-DL-alanineN-benzoyl-DL-leucineN-benzoyl-DL-valine,piperidino-p-xylene dihydrobromide-bis3-(N-benzyl-N-methylc

42、arbamoyl)-Berabrost sodiumBetaxololN-t-BOC-D,L-valineBumadizonBunololBupivacaineBupranololBupropionCarazololCarbuterolCarprofenCarvediolCathinonecis-trans-CavintonChlophedianolChlortalidoneCimetidine sulphoxideCitalopramClenbuterolCloperastineCyamemazineCyclopentolateCyclophosphamideCyklandelateDans

43、yl-DL-valine1-Decyl-3-(N,N-diehtylcarbamoyl) piperi-dine Hydrabromide2-(2,4-Dichlorophenoxy)-propionic acidDihydrodiazepam2-(4,5-dihydro-1H-imidazol-2-yl)-2-propyl-1,2,3,4-tetrahydropyrrolo 3,2,1-hi-indoleCHIRAL-AGPCHIRAL-AGPCHIRAL-CBHCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRA

44、L-HSACHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-CBHCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-CBHCHIRAL-AGPCHIRAL-CBHCHIRAL-CBHCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-CBHCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRA

45、L-AGPCHIRAL-AGP111111111111121212121212121212121212131313131313131313131313141414141414141414141415514, 29, 76, 101, 1498, 9, 12, 13, 25, 691277, 12, 131018266, 9176, 87, 149321191, 2, 7, 9, 11, 12, 13, 22,37, 38, 44, 71, 141, 15476, 10110176, 101100701451011011381408212563DihydropyridinesDiltiazemD

46、imethindeneDiperodonDisopyramideDixyrazineN-2,4-DNP-DL-N-2,4-DNP-DL-amino-n butyric acidN-2,4-DNP-DL-citrulline-amino-n-butyric acidN-2,4-DNP-DL-ethionineN-2,4-DNP-DL-glutamic acidN-2,4-DNP-DL-methionineN-2,4-DNP-DL-methionineN-2,4-DNP-DL-norleucineDobutamineDoxazosinDropropizineEpanololEphedrineEpi

47、batidineEpinephrineEtodolacFelodipineFendilineFeneterolFenoprofenFlurbiprofenFluoxetineFolinic acid (Leucovorin)H 174/48H 201/68H 309/40H 310/83HesperitinHexobarbitalHippuryl-phenyllactic acidHMG-CoA reductase inhibitorHydroxychloroquine3-Hydroxymethyl-2-methyl-9-phenyl-7H-8,9-dihydropyrano2,3-c-imi

48、dazo1,2-apyridineE-10-Hydroxy nortriptyline2-(p-Hydroxyphenoxy)propionic acid4-HydroxypropranololIbuprofenIfosfamideIsopropylidenglycerol-4-methylesterIsradipineKetamineKetoconazoleKetoprofenKetoprofen8CHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-HSACHIRAL-HSACHIRAL-A

49、GPCHIRAL-HSACHIRAL-AGPCHIRAL-HSACHIRAL-AGPCHIRAL-CBHCHIRAL-AGPCHIRAL-CBHCHIRAL-CBHCHIRAL-AGPCHIRAL-AGPCHIRAL-CBHCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-CBHCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-HSACHIRAL-CBHCHIRAL-CBHCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-A

50、GPCHIRAL-AGPCHIRAL-CBHCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-HSACHIRAL-AGPCHIRAL-HSA1515151515151515151515161616161616161616161616171717171717171727271717171718271818181818181818181919148369, 12, 13, 112, 1389, 12, 13, 1011, 2, 3, 4, 7, 9, 12, 13, 15,16, 35, 71, 85, 10129, 1018, 12

51、, 13418, 9, 12, 1481018, 32, 100, 11032, 58, 72, 77, 96, 100,110, 1131471477, 12, 13, 287862, 89, 1201477, 8, 12, 13, 29, 32, 42, 43,46, 53, 72, 96, 100, 103,110, 150, 1511112, 12, 13, 71, 1427, 12, 13, 32, 96, 100, 110,131KynurenineLaudanosineLuciferinMedetomidineMefloquineMephenytoinM

52、epivacaineMepenzolate bromideMeptazinolMetanephrineMethadoneo-Methoxymandelic acid1-(p-Methoxyphenyl)-3-butylamine-Methoxyphenylacetic acid3-Methylethylether-2-methyl-9-phenyl-7H-8,9-dihydropyrano2,3-c-imidazo1,2-apyridineMethylphenobarbitalMethylphenylcyanoacetic acid ethyl esterMetolazoneMetoprolo

53、lMianserinMidodrineModafinilMoprololMosapride1-(1-Naphthyl)-ethylamineNaproxenNefopamNicotineNitrendipineNorepinephrineNorketaminNormetanephrineOctopamineOmeprazoleOxamniquineOxazolineOxfendazoleOxodipineOxprenololOxybutyninOxyphencyclimineOxyphenoniumPamatololPargyline N-oxidePenthiobarbitalPentoba

54、rbitonePheniramineCHIRAL-HSACHIRAL-CBHCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-CBHCHIRAL-AGPCHIRAL-HSACHIRAL-HSACHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-CBHCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-CBHCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCH

55、IRAL-AGPCHIRAL-AGPCHIRAL-CBHCHIRAL-AGPCHIRAL-CBHCHIRAL-CBHCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-CBHCHIRAL-AGPCHIRAL-AGPCHIRAL-CBHCHIRAL-AGPCHIRAL-AGPCHIRAL-AGPCHIRAL-AGP19191919191919191919202020202720202020202020212121212121212121212122222222222222222222222223852661, 10

56、7281, 2, 9, 11, 12, 13, 37, 71,1411, 2, 8, 9, 12, 131269, 12, 13, 51, 84, 105, 115,143, 146147286, 8, 9, 12, 13, 20, 21, 22,23, 29, 69, 76, 87, 101,112, 149130, 13275134, 1527, 8, 12, 13, 32, 33, 49, 67,80, 100, 11710193142133,14431, 34471186, 9, 12, 13, 29, 76, 101,1121531, 9, 12, 1348128101, 112,

57、1382-Phenoxypropionic acid2-Phenylbutyric acidPhenylethanolamine2-Phenylpropionic acid (Hydratropic acid)PhenyramidolPindolol3-PPPPractololPrilocaineProcyclidineProglumidePromethazinePropafenonePropranololProxyphyllineProzacRemoxiprideRosmarinic acidSalbutamolSalmeterolSecobarbitalSolketal tosylateS

58、otalolSulfinpyrazonSuprofenTalinololTerbutalineTerodiline1,2,3,4-tetrahydro-1-naphtholTetrahydropapaverolineTetrahydrozolineTetramisoleThalidomideThiopentoneThioridazine sulfoxideTiaprofenic acidTimololTiprenololTofisopamTolamololToliprololTolperisoneTrihexyphenidylTrimipramineTropicamideUxepamVamicamideVerapamilWarfa