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Decades ofExperience and

Innovation inAnalytical

Chemistry

SolidPhaseExtraction

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Appendices

Solid Phase Extraction

For nearly 100 years MACHEREY-NAGEL has been dedicated to products for filtra-tion, water analysis and chromatography. Due to this long tradition we have beenstrongly involved in the development of chromatography and always one of the

pioneers.Since we introduced CHROMABOND in 1987 we developed the widest range ofphases and products for solid phase extraction based on silica and polymer mate-rials. These build a perfect match with all our products for HPLC, GC and TLC andoffer solutions for the whole process of analytics. Aside from all SPE standard prod-ucts we are specialised on custom made solutions for any analytical problem.

This application guide is based on our own experiences in sample preparationand applications which were kindly provided or published by our customers. Weare always interested in ongoing research and developments, and are more than

happy to expand this database also with your results and applications. We thankeveryone who contributed to this guide. Please feel free to contact us for anyquestions, ideas, technical requests or feedback.

www.mn-net.com


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Solid Phase Extraction

Introduction

Principles of solid phase extraction. . . . . . . . . . . 2Molecular interactions. . . . . . . . . . . . . . . 5

MN phases for SPE

Summary . . . . . . . . . . . . . . . . . . 8

Selection guide . . . . . . . . . . . . . . . . . . 11

Standard phases . . . . . . . . . . . . . . . . 12

Phases for special applications . . . . . . . . . . . . 19

Method development kits . . . . . . . . . . . . . 24MN products for SPE

CHROMABONDand CHROMAFIXcolumn hardware . . . . . 26

CHROMAFILsyringe filters . . . . . . . . . . . . . 29

Procedures and method development . . . . . . . . . . 32

Standard protocols for CHROMABOND . . . . . . . . . . 34

Handling of CHROMABONDand CHROMAFIXproducts . . . . 36

Accessories vacuum manifolds . . . . . . . . . . . 38Application Gallery

Biological samples and natural compounds . . . . . . . . 42

Pharmaceuticals and drugs . . . . . . . . . . . . . 69

Food and beverages. . . . . . . . . . . . . . . . 110

Environmental samples and pollutants . . . . . . . . . . 139

Appendices

Substance index . . . . . . . . . . . . . . . . . 206

Structure index (index of molecular formulas) . . . . . . . 239

CHROMABONDSPE service . . . . . . . . . . . . . 243

Contents


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Solid phase extraction (SPE) is a pow-erful method for sample preparationand is used by most chromatographers

today. It has capabilities in a broadrange of applications such as environ-mental analyses, pharmaceutical andbiochemical analyses, organic chemis-try and food analyses.

The advantages of SPE compared toclassical liquid-liquid extraction arethe low solvent consumption, theenormous time saving and the po-tential for automation. Additionally, asample preparation task can often besolved more specifically by using SPE,since different interactions of the an-alyte with the solid phase (adsorbent)are possible, and methods can be op-timised by adjusting chromatographicconditions. SPE offers a multitude of

adsorbents for polar, hydrophobicand/or ionic interactions, while liquid-liquid extraction is limited to partition

equilibriums in the liquid phase.The CHROMABOND columns andCHROMAFIX cartridges from MNwhich have been developed especiallyfor SPE provide rapid, economical andeffective systems for sample prepa-ration. They can be used to processsamples for HPLC, GC, TLC, UV or IRspectroscopy and many more. For the

increasingly sensitive chromatograph-ic analyses good sample preparationis essential, because it protects thechromatographic columns, and it al-lows a greater sensitivity by removal ofinterfering matrix components. A se-lective and specific sample preparationthus is a prerequisite for reasonable,economical and sensitive analyses.

The main objectives of SPE are removalof interfering matrix components andselective concentration and isolation ofthe analytes. Enrichment can increasethe detection sensitivity by a factor of100 to 5000. Often this step is neces-sary to reach the concentration limit ofdetection for the analytes of interestfor qualitative or quantitative analyses,i.e. without enrichment often a reliable

analysis at trace level is not possible.In order to maintain the high qual-ity of our CHROMABOND columnsand CHROMAFIX cartridges and thusto ensure reproducible extraction re-sults, CHROMABOND adsorbents aresubject to defined, stringent controlcriteria. Our quality control from thestarting materials through all inter-

mediate steps up to the final productguarantees consistent quality.

Principles of solid phase extraction


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In general, SPE can be used for threeimportant purposes in up-to-dateanalyses:

concentration of the analyte removal of interfering substances

changing the matrix of the analyteas needed for subsequent analyses

In most cases these three effects occurtogether. Since analytes can be eitheradsorbed on the SPE packing mate-

rial or directly flow through while theinterfering substances are retained,two general separation procedures arepossible. The first case is shown in thefigure below.

Retention of the analyte

Introduction


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The sample is pressed or drawnthrough the solid phase, and the an-alyte molecules are enriched on the

adsorbent. Interfering componentsand solvent molecules (matrix) arenot retained. Then remaining interfer-ing components are washed from theadsorbent with a suitable washing so-lution. Finally, the analyte is removedfrom the adsorbent by elution with asuitable solvent.

The considerations made above indi-cate that an optimum SPE presents apoor column-chromatographic sepa-

ration. If in chromatography substanc-es run at the solvent front, or if sub-stances are adsorbed at the columnhead, effi cient chromatography is notpossible using one eluent; one can callthis digital chromatography.

Retention of interfering components

In some cases other interfering com-ponents may remain on the adsorbent.

Such a strong adsorption of interferingcomponents offers another possibilityfor the prepurification of diffi cult ma-trices, such as waste oils or sludge. Ifthe analytes show no interaction withthe adsorbent and if only the interfer-ing components are retained, the solidphase can be used to simply filter thesample, as shown in the figure above.

An adsorbed substance can be re-moved from the adsorbent by a step-

wise increase of the elution strength ofthe eluent (step gradient technique).

However, this method can be utilisedeffi ciently for a preseparation of groupsof compounds or a single analyte fromthe matrix. It is extensively used forclean-up by solid phase extraction.



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Nonpolar interactionsoccur betweenhydrocarbon residues of the functionalgroups of the adsorbent and the an-

alyte. Since most organic compoundshave a nonpolar structure, they can beadsorbed to nonpolar adsorbents viavan-der-Waals forces.

Almost all organic compounds have acertain potential for nonpolar interac-tions. Exceptions are compounds pos-sessing a large number of polar or evenionic groups which shield the nonpolar

character of the carbon skeleton (e.g.carbohydrates).

Unmodified silica on the other handshows no nonpolar interactions. Sincethe functional groups of most modified

silicas are bonded to the silica surfacevia a hydrocarbon spacer, these modi-fied silicas show a certain degree ofnonpolar interactions.

Typical adsorbents with a pronouncednonpolar character are e.g. C18 ec,C18, C18 Hydra and C8 modified sili-cas. They show a seemingly low selec-tivity, because their functional groups,

the alkyl substituents, can interact withalmost all nonpolar analytes. This canbe used for the isolation of substancegroups of different structure.

Nonpolar interactions on C18 silica

Retention Elution

nonpolar (relative)

polar (relative)

Molecular interactions


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Polar interactions include hydrogenbonds, dipole-dipole and -interac-tions, which can occur between many

different adsorbents and functionalgroups of the analytes. Some of theseinteractions are possible between ami-no, hydroxyl and carbonyl groups aswell as aromatic rings, double bondsand groups with hetero-atoms suchas nitrogen, sulphur, phosphorus andoxygen. Typical adsorbents for polarinteractions are unmodified silica, CN,NH2and OH (diol) modified silicas.

It should be noted, that in SPE the in-teractions described in this chapter arenot found in pure form, but in combi-

nation. For example, modified silicas,unless they have been subjected toendcapping (silanisation of residual si-lanol groups with short-chain silanes),still possess free silanol groups, whichcan enter into secondary interactions.

Polar interactions on silica

Retention Elution

In general, polar compounds are easilyadsorbed to a polar adsorbent from anonpolar environment and are elutedwith a polar solvent. The oppositeholds true for nonpolar compounds.They are easily adsorbed from a polarenvironment onto nonpolar surfaces.

Elution is achieved by solvents of lowerpolarity.

nonpolar (relative)

polar (relative)



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Ionic interactions occur betweencharged analytes and an adsorbentwith a functional group of opposite

charge. Cationic groups are present inprimary, secondary, tertiary and qua-ternary amines and inorganic cations,e.g. calcium, sodium, magnesium etc.Examples for anionic groups are carb-oxylic and sulphonic acids, phosphatesand similar groups.

Retention via these ion exchange in-teractions is enhanced in a matrix oflow ionic strength and a counter ion

of low selectivity (e.g. acetate, Na+

).For elution a solvent with high ionicstrength and high selectivity is pre-ferred (e.g. citrate or Ca2+).

Ionic interactions on SA silica (cation exchange)

Retention Elution

Ionic interactions on SB silica (anion exchange)

Retention Elution

Molecular interactions


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Selectivity and capacitySelectivity is the ability of an adsorb-ent to discriminate between the an-

alyte and other sample components.Thus, in general it describes the abilityof the solid phase to adsorb the an-alyte, while the undesired componentsare not retained.

The selectivity depends on the chemi-cal structure of the analyte, the prop-erties of the adsorbent, the compo-sition of the sample matrix and the

eluent used. Optimum selectivity isachieved via functional groups of theanalyte, which are not present in thesample matrix and other interferingcomponents.

The capacity of an adsorbent is de-fined as the total amount of an analyte,which is adsorbed to a known amountof the adsorbent under optimum con-

ditions. For ion exchangers the capac-ity is usually given as milliequivalentsper gram [meq/g]. For other silica ad-sorbents capacity values are about 3 5 % of the amount of adsorbent. Thepolystyrene-divinylbenzene based ad-sorbent resins HR-P and Easy featurean exceptionally high capacity of 30 %.Thus, if the capacity and the amount ofanalyte to be concentrated are known,

one can roughly estimate the amountof adsorbent required.

Summary of SPE phasesFor solid phase extraction MN offersadsorbents based on polymer res-ins (Easy, HR-P), surface-modifiedsilica materials as well as Florisil,polyamide, and aluminium oxide. Spe-cial phases for defined applications inpharmaceutics, environmental analysisand ion chromatography complete theprogramme. The following pages de-scribe the properties of all availablephases.

Surface-modified silicas are stable inthe pH range of about 2 to 8; however,in practical SPE they can often be usedin a wider pH range, since cleavage ofthe functional groups is a function oftime, and the adsorbents usually haveonly short-time contact with the sol-vents. In addition, the modified silicasare stable in almost all organic sol-vents. Silicas are hard materials, whichshow neither swelling nor shrinking,contrary to polyamide and the adsorb-ent resins HR-P and Easy. They havea mean pore size of 60 , which al-lows adsorption of compounds withmolecular weights up to about 5000.Larger molecules can not diffuse intothe pores and are thus not retained.

In addition to our standard phasesthe

MN programme also comprises spe-cial and combination phasesfor SPE.As an example we want to mentionthe solid phase extraction of polycy-clic aromatic hydrocarbons (PAH) andpolychlorinated biphenyls (PCB).

Especially for extraction of the 16 PAHacc. to EPA from soil samples we havedeveloped the combination phase CN/

SiOH. This combination utilises theability of the cyanopropyl phase to

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selectively adsorb polycyclic aromat-ics. For isolation of the 16 PAH acc.to EPA from water our special column

CHROMABOND

C18 PAHis especiallysuited, reproducibly allowing recoveryrates up to 97%. The CHROMABONDNH2/ C18combination column is analternative for the enrichment of PAHespecially from humic acid-rich water.The NH2phase allows removal of theinterfering humic acids.

The column CHROMABOND SiOH-

H2SO4/SAhas been developed for theextraction of PCB from oil accordingto the German industrial standard DIN51 527. It contains an upper layer ofsilica impregnated with H2SO4 and alower layer of a strongly acidic cationexchanger with benzenesulphonic acidgroups. This column is used togetherwith the silica column CHROMABONDSiOH. Both columns are available as

Kombi-Kit PCB (Cat. No. 730125). Inaddition to the DIN procedure thereis a method, which is especially rec-ommended for the enrichment of PCBfrom waste oil samples. The combina-tion phase SA/SiOH, which has beendeveloped for this purpose, consists ofa cation exchanger with benzenesul-phonic acid groups as upper layer and

silica as lower layer.For PCB determination from sludgeacc. to the German sludge regulationswe have developed the special columnCHROMABOND NAN. The columnpacking is a combination of Na2SO4and silica impregnated with AgNO3.

One of the most important applicationsfor CHROMABONDand CHROMAFIXPS phases for ion chromatography is

the removal of overlapping compo-nents. Elimination of interfering com-ponents can on the one hand improvethe chromatographic separation, if theinterfering components overlap theanalytes in the chromatogram, on theother hand it also improves lifetime ofthe chromatographic column, since in-terfering components can irreversiblycover the column packing. In addition

to this simple clean-up of aqueoussamples, however, the cartridges canalso be used for an enrichment of theanalytes.

CHROMABOND and CHROMAFIX PSphases are offered in several differentchemical modifications. Base materialin all cases is a polystyrene-divinyl-benzene copolymer of highest purity,

which features a very low degree ofswelling and is thus very well suitedfor chromatography. This base mate-rial allows a reliable function of thecartridges over the whole pH range.The mean pore diameter of this mate-rial is 100 , the mean particle size is100 m. The most important fields ofapplication for PS-RP, PS-OH, PS-H+,

PS-Ag+

and PS-Ba2+

reach from elimi-nation of nonpolar constituents to theremoval of specific polar components.

Summary


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Typical solvents for SPE

Polarity Solvent Miscibility with water

nonpolar Hexane no

Isooctane no

Petroleum ether no

Cyclohexane no

Carbon tetrachloride no

Chloroform no

Methylene chloride no

Tetrahydrofuran yes

Diethyl ether no

Ethyl acetate poor

Acetone yes

Acetonitrile yes

Isopropanol yes

Methanol yesWater yes

polar Acetic acid yes

Selection of SPE phasesThe scheme on the following page is meant as a first guide to the applicationof CHROMABOND columns or CHROMAFIX cartridges and considers mostly

primary interactions. It is only valid for samples with molecular weights below5000 Dalton to avoid size exclusion effects which might block the pores of theSPE silica.

The solvents recommended for elution should also be considered as a first se-lection. Applicability of other solvents or solvent mixtures is determined by thepolarity required for a separation.

MN phases for SPE


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Selection guide for SPE phases and solvents

Sample

solubility

Solvent Sample

polarity

Phases recommended

for adsorption

Solvents recom-

mended forelution (selection)

soluble inwater

notionic aqueous

nonpolar

Easy, HR-PC18 ec, C18, C18 HydraC8, C4, C2, C6H5CN

hexaneCH2Cl2acetonitrilealcohols

moderatelypolar

SiOHNH

2

CHCl3, CH2Cl2ethyl acetatealcoholswater

polar

CN, OHPADMANH2

CHCl3,CH2Cl2ethyl acetatealcoholswater

ionic aqueous

cationic

PCASA

PSAPS-H+

acidssalt solutions

buffers

anionic

SBNH2DMAPS-OH

acidssalt solutionsbuffers

soluble inorganicsolvents

aqueous nonpolar

Easy, HR-PC18 ec, C18, C18 Hydra

C8, C4, C2, C6H5CNPS-RP

hexaneCH2Cl2

acetonitrilealcohols

organicmoderately

polarSiOHNH2

CHCl3, CH2Cl2ethyl acetatealcohols

organic polar

CN, OHPA

DMANH2

CHCl3, CH2Cl2ethyl acetate

alcohols

Summary


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The large range of analytes to be iso-lated and the great variability in sam-ple matrices require a large number

of phases for solid phase extrac-tion. Combined with different column

hardware for different sample sizes,this results in a considerable diversifi-cation of our programme for SPE. The

following pages provide a brief de-scription of all MN phases for SPE.

Easy polar, bifunctionally modifiedpolystyrene-divinylbenzene adsorbent resin

polar modified polystyrene-divinylbenzenecopolymer with a weak ion exchanger

specific surface 650 700 m2/g,particle size 80 m, pore size 50 ,pH stability 1 14

due to bifunctional modification much more hy-drophilic than conventional polystyrene-divinyl-benzene polymers and thus easily wettable withwater

recommended applications:

drug analysis from urine,blood, serum, plasmapharmaceuticals / activeingredients from tablets,creamspolar herbicides / pesti-cides from water (acidic,neutral, basic)polar phenols from waterpolyaromatic compoundspolychlorated biphenyls

HR-P polystyrene-divinylbenzene adsorbent resinhighly porous polystyrene-divinylbenzenecopolymer

specific surface 1 200 m2/g,particle size 50 100 m

very high binding capacity, up to 30% of adsorb-ent weight (for comparison: silica adsorbentsabout 3%)


aromatic compoundsphenols from waternitroaromatics from waterpesticides from waterPAH from oil

C18 / C18 f (f = fast flow) octadecyl silicabase material silica, pore size 60 , particle size45 m for C18, 100 m for C18 f (for fast flow),specific surface 500 m2/g, pH stability 2 8

octadecyl phases, not endcapped, carbon content 14%

similar to C18 ec, however possesses more freesilanols (SiOH), which allow secondary interac-tions with polar groups of the analytes


nonpolar compoundspesticides

C18 f for viscous samples

MN phases for SPE


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C18 ec / C18 ec f (f = fast flow) octadecyl silica, endcappedbase material silica, pore size 60 ,

particle size 45 m for C18 ec, 100 m forC18 ec f (for fast flow),specific surface 500 m2/g,pH stability 2 8

octadecyl phases, endcapped,carbon content 14%

very nonpolar, hydrophobic interactions with awide variety of organic compounds

advantageous for clean-up of samples with largestructural variations (polarity differences)


nonpolar compoundsaflatoxins, amphetamines,antibiotics, antiepilepticsbarbiturates, caffeine,drugs, preservativesfatty acids, nicotine, PAH,pesticides, PCBheavy metals, vitamins

very well suited for desalt-ing of samples

C18 ec f for viscous sam-ples

C18 Hydra octadecyl silica for polar analytesbase material silica, pore size 60 ,particle size 45 m, specific surface 500 m2/g,pH stability 2 8

special octadecyl phase for polar analytes, notendcapped, carbon content 15%


more polar compounds likepesticides and their polardegradation productsphenolsphenoxycarboxylic acidsnitroaromaticspharmaceuticals

C8 octyl silicabase material silica, pore size 60 ,

particle size 45 m, specific surface 500 m2

/g,pH stability 2 8

octyl phase, not endcapped, carbon content 8%

similar to C18, however slightly more polar

secondary interactions with polar compounds aremore pronounced due to shorter alkyl chains


pesticidesPCB

Standard phases


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C4 butyl silicabase material silica, pore size 60 ,


/g,pH stability 2 8

butyl phase, not endcapped, carbon content 7%

slightly more polar than C18 or C8

due to shorter alkyl chains the silica surface isnot completely shielded


compounds, which are toostrongly retained on C18 orC8e.g. analgetics from blood

C2 dimethyl silicabase material silica, pore size 60 ,particle size 45 m, specific surface 500 m2/g,pH stability 2 8

dimethyl phase, not endcapped,carbon content 4%

similar to C4


e.g. antiepileptics fromplasma

C6H11ec cyclohexyl silica, endcappedbase material silica, pore size 60 ,particle size 45 m, specific surface 500 m2/g,pH stability 2 8

cyclohexyl phase, endcapped, carbon content 9%

alternative phase for the mid-polar range


phenols from waterchloroanilines from wastewateranthelmintics from tissue

C6H

5 phenyl silica

base material silica, pore size 60 ,particle size 45 m, specific surface 500 m2/g,pH stability 2 8

phenyl phase, carbon content 8%

polarity similar to C8

in addition to hydrophobic interactions more se-lective adsorption is possible by -interactionsdue to the electron density of the phenyl ring


aflatoxinscaffeinephenols

MN phases for SPE


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NO2 nitrophenyl silicabase material silica, pore size 60 ,


/g,pH stability 2 8

nitrophenyl phase, carbon content 5.5%


aromatics

NH2 aminopropyl silicabase material silica, pore size 60 ,particle size 45 m, specific surface 500 m2/g,pH stability 2 8

aminopropyl phase, carbon content 3.5%

polar, weak anion exchanger


trace elementslipids

DMA dimethylaminopropyl silicabase material silica, pore size 60 ,particle size 45 m, specific surface 500 m2/g,pH stability 2 8

dimethylaminopropyl phase, carbon content 3.5%polar, weak anion exchanger


similar to NH2 slightlyweaker anion exchanger

CN cyanopropyl silicabase material silica, pore size 60 ,particle size 45 m, specific surface 500 m2/g,pH stability 2 8

cyanopropyl phase, carbon content 5.5%polar to mid-polar

in addition to weak hydrophobic interactions se-lective interactions are possible due to the highelectron density of the CN group


cyclosporinscarbohydrates

Standard phases


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OH diol silicabase material silica, pore size 60 ,


/g,pH stability 2 8

diol phase, carbon content 5.5%

polar

properties similar to SiOH


antibioticsprostaglandins

SiOH unmodified silica

unmodified, weakly acidic silica, pore size 60 ,particle size 45 m, specific surface 500 m2/g,pH stability 2 8

very polar

adsorbs humidity from air, for this reason itshould be kept well closed and if necessary driedbefore use

due to its high affi nity for polar compounds itshould not be conditioned with polar (e. g. meth-anol) or water-containing solvents

recommended applications: aflatoxins

chloramphenicolpesticidessteroidsvitamins

Alox A / Alox N / Alox B aluminum oxide, acidic, neutral, basicaluminium oxide, high purity, pore volume0.90 ml/g, particle size 60 150 m, specificsurface 150 m2/g


together with phase SA forPCB and pesticides

Properties of the individual modifications:

Alox A: aluminium oxide, acidic pH value 4 0.3

Alox N: aluminium oxide, neutral pH value 7 0.5

Alox B: aluminium oxide, basic pH value 9.5 0.3

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Florisil magnesium silicatematrix magnesium silicate (MgO-SiOH 15:85),

high purity, particle size 150 250 m Florisilis a product and registered trademark of

Floridin Company, USA


organic tin compoundsaliphatic carboxylic acidsPCB, PAH

PA polyamide 6matrix polyamide 6, unmodified, high purity,particle size 40 80 m


flavonoidsPAH

PCA propylcarboxylic acid cation exchanger based on silicabase material silica, pore size 60 ,particle size 45 m, specific surface 500 m2/g,pH stability 2 8

propylcarboxylic acid modified silica

weakly acidic cation exchanger


strong cations

SA benzenesulphonic acid cation exchanger based on silicabase material silica, pore size 60 ,particle size 45 m, specific surface 500 m2/g,pH stability 2 8

benzenesulphonic acid modified silica

strongly acidic cation exchanger

capacity ~ 0.5 meq/g adsorbent with hydrophobic and -interactions

(benzene ring)

ion exchange of organic compounds from aque-ous matrix

elution of interesting compounds with solventsystems, which compensate the ionic and nonpo-lar interactions, e.g. methanolic HCl


amino acidschlorophyllPCB

Standard phases


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PSA propylsulphonic acid cation exchanger based on silicabase material silica, pore size 60 ,


/g,pH stability 2 8

propylsulphonic acid modified silica

very strong cation exchangercapacity ~ 0.7 meq/g

contrary to the SA phase no -interactions


weak cations

SB quaternary ammonium anion exchanger based on silicabase material silica, pore size 60 ,particle size 45 m, specific surface 500 m2/g,pH stability 2 8

silica modified with quaternary amine

strongly basic anion exchangercapacity ~ 0.3 meq/g

not suited for very strong anions such as sul-phonic acids, because these are diffi cult to elute


organic acidscaffeinesaccharin

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PS-RP / PS-OH/ PS-H+ phases for RP / ion chromatographyPS-Ag+/ PS-Ba2+

base material silica, pore size 60 ,particle size 45 m,specific surface 500 m2/g,pH stability 2 8

silica modified with quaternary amine

strongly basic anion exchangercapacity ~ 0.3 meq/g

not suited for very strong anions such assulphonic acids, because these are diffi cult

to elute

recommended applications:removal of interfering compounds

improves chromatographic separa-tion, if the interfering componentsoverlap with the analyte in thechromatogram

improves lifetime of the chroma-tographic column, since interferingcomponents can irreversibly blockthe column packing

enrichment of the analytes

Properties of the individual modifications:

PS-RP hydrophobic PS/DVB copolymer removal of organic interfering com-ponents from water

PS-OH strong PS/DVB anion exchanger,OHform, capacity 0.6 meq/g

removal or concentration of anionsfrom water

increasing the pH value in acidicsamples

PS-H+

strong PS/DVB cation exchanger,H+form, capacity 2.9 meq/g removal or concentration of cationsfrom water

decreasing the pH value of basicsamples

PS-Ag+ strong PS/DVB cation exchanger,Ag+form

removal of halide ions from water

PS-Ba2+ strong PS/DVB cation exchanger,Ba2+form

removal of sulphate ions from water

Diamino special phase for determination of pesticides in foodsilica with primary and secondary aminefunction

removes polar compounds (e.g. organicacids, pigments, sugars) from matrices likefruit or vegetables with low fat content


quick and cheap determination ofpesticides in strong matrix con-taminated samples by GC (QuEChERSmethod = Quick Easy Cheap Effec-tive Rugged Safe)

Phases for special applications


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ABC18special phase for analysis of acrylamide in food

octadecyl silica phase with ion exchange func-tions for acrylamide analysis

recommended applications: clean-up of acrylamide

from ultra-heated starch-containing food, such aspotato chips and othersnacks, french fries, crisp-bread, cereals etc.

Important note:

Miniumum concentration of acrylamide should be 70 g/kg.

The procedure includes no concentration step.Acrylamide and the isotopically labelled form, is carcinogenic, mutagenic and neu-rotoxic.

Acrylamide is created at temperatures above 100 C from sugar and proteins, e.g.from potatoes or grain during the process of frying, baking, roasting or grilling.The formation depends on temperature, starting at 120 C and increasing with moreelevated temperatures. In cooked food, no acrylamide is found. During the processof baking or frying, however, a remarkable amount of acrylamide can be formed.

Drug special silica phase for drug analysisbase material silica, pore size 60 ,particle size 45 m, specific surface 500 m2/g,pH stability 2 8

special bifunctional modification


enrichment of acidic, neutraland basic drugs from urineor plasma

Tetracycline special phase for enrichment of tetracyclinessilica phase with special C18 modification, testedfor tetracyclines

constant recovery rates for the title compounds(every batch individually tested)


tetracyclines from biologicalsamples

MN phases for SPE


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Crosslinksspecial phase for enrichment of collagen crosslinks

special cellulose phase for enrichment of colla-gen crosslinks

recommended applications: collagen crosslinks in urine

Pyridinoline and deoxypyridinoline are collagen crosslinks occuring in bones and car-tilage. If these substances are released, they can be detected in the urine. In cases ofincreased bone catabolism (e. g. during osteoporosis) the urine concentrations of py-ridinoline and deoxypyridinoline are increased.

AOX AOX from waters with high salt loads (DIN 38409 H22)special PS-DVB phase recommended applications:

extraction of AOX fromwaters containing high saltloads / organic pollutants inaccordance with DIN 38409 H22

CN/SiOH combination phase for PAH analysisspecial combination phase:

cyanopropyl phase for selective adsorption ofpolycyclic aromatics via -interactions

unmodified silica phase for removal of polarcompounds


extraction of the 16 PAHaccording to EPA from soilsamples

C18 PAH octadecyl silica for PAH analysisbase material silica, pore size 60 ,particle size 45 m, specific surface 500 m2/g,pH stability 2 8

special octadecyl modification for enrichment ofPAH, not endcapped, carbon content 14%


PAH from water



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NH2/C18 combination phase for PAH analysisspecial combination phase:

aminopropyl phase for removal of interfering hu-mic acids

octadecyl phase for enrichment of PAH


PAH from water containinghumic acids

Na2SO4/Florisil hydrocarbons from water

acc. to DIN H-53 / ISO DIS 9377-4

special combination phase of sodium sulphate

and Florisil


hydrocarbons from drinking,surface and waste waters

SiOH-H+/SA combination phase for PCB analysisspecial combination phase:

SiOH-H+: H2SO4-impregnated silica phase foroxidation of accompanying compounds to ionicand/or polar compounds

SA: strongly acidic cation exchanger based onsilica with benzenesulphonic acid modificationfor removal of ionic and sulphur-containingcompounds

This combination column is used together witha SiOH column. Both columns together are avail-able as Kombi-Kit PCB.


extraction of PCB from oilwith reference to German in-dustrial standard DIN 51527,part 1

SA/SiOH combination phase for PCB analysisspecial combination phase:

SA: strongly acidic cation exchanger based onsilica with benzenesulphonic acid modification

SiOH: unmodified silica for removal of polar com-pounds


extraction of PCB fromwaste oil (hexane extract)

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NAN special phase for PCB analysisspecial combination phase:

SiOH/AgNO3phase for removal of sulphur, sul-phur-containing and polar compounds

sodium sulphate for removal of trace water


extraction of PCB fromsludge

DNPH special phase for enrichment of carbonyl compoundssilica impregnated with 2,4-dinitrophenylhydra-zine (DNPH)

capacity about 75 g formaldehyde, workingrange 1 5000 ppb

samples can be passed through the cartridge inboth directions

each cartridge is sealed in a laminated aluminiumbag

carbonyl compounds adsorbed as 2,4-dinitro-phenylhydrazine derivatives (hydrazones) can beeluted from the cartridges with acetonitrile


carbonyl compounds (alde-

hydes and ketones) from air for enrichment aspirate air

through the cartridge withmax. 2 l/min

Dry special phase for drying of organic samplesanhydrous high-purity sodium sulphate whichforms Glaubers salt with traces of water

for removal of larger quantities of water severalcartridges can be combined in series


removal of traces of waterfrom organic solutions



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CHROMABONDand CHROMAFIXmethod development kitsAll individual CHROMABONDand CHROMA-BONDLV columns as well as all CHROMA-FIX cartridges are sealed in units of fivecolumns each to prevent adsorption ofcontaminants from the environment, e. g.laboratory air.

Only CHROMAFIXDNPH cartridges are indi-vidually sealed in laminated aluminium bags.If you do not know the behaviour of yoursamples in solid phase extraction, or if youwant to optimise the extraction procedure, werecommend our method development kits.

SPE method development kits ordering information

Designation Contents of the kit Cat. No.

Investigating the best separation mechanism for a clean-up procedureCHROMABONDstandarddevelopment kit

10 columns each with 1 ml / 100 mg:C18, C18 ec, C8, Phenyl, NH2, DMA, OH, CN,SiOH, SA, SB

730110

Selecting the optimum RP phase for a clean-up procedure

CHROMABONDRPdevelopment kit I

10 columns each with 3 ml / 500 mg:C18, C18 ec, C8, C4 and 10 columns with3 ml/200 mg HR-P

730197

CHROMABONDRP

development kit II

10 columns each with 1 ml / 100 mg:

C18, C18 ec, C8, C4 and HR-P

730207

CHROMAFIXRPdevelopment kit I

10 cartridges each CHROMAFIXS:C18, C18 ec, C8, C 4 and HR-P

731883

CHROMABONDRPdevelopment kit III

10 columns each with 3 ml / 500 mg:C18, C18 ec, C18 Hydra, C8 and10 columns with 3 ml / 200 mg HR-P

730490

CHROMABONDRPdevelopment kit IV

10 columns each with 1 ml / 100 mg:C18, C18 ec, C18 Hydra, C8 and HR-P

730491

CHROMAFIXRPdevelopment kit II

10 cartridges each CHROMAFIXS:C18, C18 ec, C18 Hydra, C8 and HR-P

731886

CHROMABONDRPdevelopment kit V

10 columns each with 3 ml / 500 mg:C6H5, NO2, C6H11ec, C4, C2

730492

CHROMABONDRPdevelopment kit VI

10 columns each with 1 ml / 100 mg:C6H5, NO2, C6H11ec, C4, C2

730493

CHROMAFIXRPdevelopment kit III

10 cartridges each CHROMAFIXS:C6H5, NO2, C6H11ec, C4, C2

731887

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Designation Contents of the kit Cat. No.

Selecting the optimum polar phase for a clean-up procedure

CHROMABOND

polardevelopment kit I 10 columns each with 3 ml / 500 mg:SiOH, Florisil, NH2, CN, OH 730199

CHROMABONDpolardevelopment kit II

10 columns each with 1 ml / 100 mg:SiOH, Florisil, NH2, CN, OH

730208

CHROMAFIXpolardevelopment kit

10 cartridges each CHROMAFIXS:SiOH, Florisil, NH2, CN, OH

731884

Selecting the optimum ion exchanger for a clean-up procedure

CHROMABONDion exchangedevelopment kit I

10 columns each with 3 ml / 500 mg:SA, SB, PS-OH, PS-H+, DMA

730206

CHROMABOND

ion exchangedevelopment kit II 10 columns each with 1 ml / 100 mg:SA, SB, PS-OH, PS-H+, DMA 730209

CHROMAFIXion exchangedevelopment kit I

10 cartridges each CHROMAFIXS:SA, SB, PS-OH, PS-H+, DMA

731885

CHROMABONDion exchangedevelopment kit III

10 columns each with 3 ml / 500 mg:SA, PSA, PCA, PS-H+

730494

CHROMABONDion exchangedevelopment kit IV

10 columns each with 1 ml / 100 mg:SA, PSA, PCA, PS-H+

730495

CHROMAFIXion exchangedevelopment kit II

10 cartridges each CHROMAFIXS:SA, PSA, PCA, PS-H+

731888

Phase selection for clean-up procedures for environmental samples

CHROMABONDkit forenvironmental samplepreparation

10 columns each with 3 ml / 200 mg HR-P,6 ml / 1000 mg C18 ec, 6 ml / 2000 mg C18PAH, 6 ml / 500/1000 mg CN/SiOH,3 ml / 500/500 mg SA/SiOH

730205

Method development kits


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CHROMABONDpolypropylene columnsThe polypropylene sample reservoiris compatible with most solvents. Theadsorbent is kept in place by two (forcombination columns with two phas-es three) polyethylene filter elements(pore size 20 m), which are chemi-cally very inert. The figure shows the3 standard sizes (scale 1:1).

CHROMABONDglass columnsIf it is necessary to exclude any influ-ence from the column material, you canorder the complete CHROMABONDprogramme in 1, 3 and 6 ml glass col-umns. CHROMABONDglass columnsare available with all phases describedon pages 12 to 23. The cataloguenumbers are differentiated by the let-ter G, e.g. 730001 (polypropylene) and730001 G (glass). The adsorbent is

kept in place by two (for combinationcolumns with two phases three) glassfibre filter elements (nominal pore size1 m), which are chemically very inert.

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CHROMABONDLV columns

These large volume columns areavailable with three different sorbentweights (100, 200 and 500 mg) andfeature a funnel-shaped reservoir with15 ml volume. Especially for clinicalsamples this offers the advantage, thatthe whole sample (e. g. urine, serum,blood) can be applied to the column inone step. Additionally, CHROMABONDLV can be directly used in the Zymate

lab robots of Zymark. The figure showsthe original size of the LV columns.

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CHROMAFIXcartridgesCHROMAFIX cartridges are manu-factured from polypropylene and al-

low application of almost all usualsolvents. The adsorbent is kept inplace by two polyethylene filter ele-ments (pore size 20 m). As adsorb-ents we use the same phases as forour CHROMABOND columns, thebasic procedure for sample prepara-tion with these cartridges from condi-tioning via sample application, wash-

ing and elution is the same as withCHROMABONDcolumns.

The CHROMAFIX programme con-sists of different sizes (S, M, and L)with different sorbent weights. Con-trary to CHROMABOND columns,CHROMAFIXcartridges have a femaleLuer tip at the inlet and a male Luertip as exit, thus these cartridges offeran alternative way of handling sam-ples in SPE. They are especially suitedfor convenient solid phase extractionof small sample volumes.

CHROMABONDMULTI 96SPE is a reliable method for samplepreparation. It is often used for en-

richment or clean-up of numeroussamples prior to analysis. For largenumbers of samples MN offers 96-well microtiter plates for SPE.

The method development time isminimal. CHROMABOND MULTI 96can be supplied with any of the MNSPE adsorbents. Methods that havebeen worked out for CHROMABOND

columns or CHROMAFIX cartridgescan be easily transferred to CHROMA-BONDMULTI 96.

Simultaneous preparation of 96 sam-ples is economical because of the sav-ing in time and solvent.

Advantages of this high-throughputsystem are:

simultaneous preparation of 96

samples; this means a 4-fold in-crease over traditional 24-positionSPE processors

use of multi-channel pipettors fa-cilitates liquid transfer steps

readily adaptable to all common au-tomated / robotic handling systems

minimised dead volume ( 40 l)

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CHROMAFIL

With CHROMAFIL, rapid purificationand removal of particles from liquid

samples or gases is very simple: justplace the filter on the syringe, and youare ready for filtration. Special ma-nipulations are not required. Contami-nation of sensitive instrumentation bysolid impurities can be avoided, thusincreasing lifetime of chromatographiccolumns an equipment.

Advantages:Polypropylene housingbetter solvent stability compared toacrylate and polystyrene filters

Shells ultrasonically sealed, not gluedno extractable components fromglues

Filtration in both directions pos-sible, the liquid cannot bypass the

membrans Luer lock on side of entrysafe connection on the high pres-sure side

Luer exitstandard luer for 3 and 25 mm fil-ters, minispike luer with low deadvolume and small OD for 15 mmfilters

Deflectorthe stream of liquid is broken anddistributed, and does not directlyhit the membane: this preventsrupture of the membrane

Star-shaped distribution devicethe liquid is evenly distributed tothe whole membrane surface: thisresults in a better utilisation of the

total area; the filter is not pluggedup rapidly; high flow effi ciency

Colour codedfilters with 0.2 m pores have ayellow upper shell, that of filters

with 0.45 m pores is colourless;the different membrane types aredistinguished by different colours

Low dead volume~120 l for 25 mm , 12 l for15 mm , 5 l for 3 mm

CHROMAFIL filters are available withpore seizes of 0.2 and 0.45 m (ex-ceptions: PET filters also 1.2 m, glass

fibre filtes only 1 m) and filter sizes of25, 15 and 3 mm diameter. The smalldiameter filters are especially recom-mended for very small samples, whichrequire extremely low dead volumes.

Recommended filter size dependingon sample volume

sample volume recommendedfilter diameter

1 ml 3 mm1 10 ml 15 mm

10 100 ml 25 mm

Technical dataThe membrane housing consists ofpolypropylene (PP). This material isvery resistant towards most solventsand has a very low content of extract-able substances. Thus it can be usedwith almost all solvents, acids andbases (see table). The special thick rimof the housing is ideal for use of thefilters in laboratory robots (e.g. Bench-mate). Filter inlet and filter exit canbe fitted to the CHROMABOND col-umns for selective sample preparationwith the aid of a special adaptor.

All filters can be autoclaved at 121 Cand 1.1 bar for 30 min.

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ApplicationDepending on your filtration problemyou can choose filter membranes made

from different materials:Cellulose mixed esters (MV) thismembrane is recommended for allfiltrations in aqueous or polar media.

Cellulose acetate(CA) this mem-brane features a very high shapestability in aqueous solutions and avery low binding capacity for pro-teins. CA filters are available in a

sterile and a nonsterile package.For sterile filtration of non-ster-ile solutions we recommend theCHROMAFIL Sterilizer. For filtra-tions under sterile conditions, theproven CHROMAFIL CA-20/25 Sand CA-45/25 S are the filters ofchoice. All CA filters feature an ex-tremly low binding capacity for pro-

teins (2.9 g/cm2

BSA). Regenerated cellulose (RC) thishydrophilic membrane features avery low adsorption; it is recom-mended for filtration of aqueousand organic/aqueous liquids

Polyamide (PA) Nylon this is arather hydrophilic membrane; itis recommended for filtration of

aqueous and organic/aqueous me-dium polar liquids.

Teflon (PTFE) a hydrophobicmembrane; ideal for filtration ofnonpolar liquids and gases. It isvery resistant towards all kinds ofsolvents as well as acids and bases.By flushing with alcohol, followedby water, the originally hydropho-

bic membrane can be made morehydrophilic.

Polyvinylidene difluoride (PVDF)with integrated glass fibre prefilter compared to PTFE, this membrane

also features hydrophilic interac-tions. It can be used for the filtra-tion of polar and nonpolar solu-tions.

Polyester (PET) this fabric-rein-forced membrane features an out-standing chemical resistance and issuited for polar as well as nonpolaresolvents. This all purpose mem-

brane is recommended for filtra-tion of aggressive media, for dustand aerosol analyses, ultrapurifica-tion of solvents etc. It is very wellsuited for TOC/DOC determination.The membrane is not cytotoxic anddoes not inhibit the growth of mi-croorganisms and higher cells.

Glass fibre(GF) The nominal poresize of these filters is 1 m. Theycan be used for solutions with highloads of particulate matter or forhighly viscous solutions (e. g. soilsamples, fermentation broths) ei-ther alone or combined with otherCHROMAFIL filters. When mem-brane filters are combinde withglass fibre filters, they preventplugging of the membrane. Used

alone, glass fibre filters allow high-er flow rates than e.g. a 0.45 mfilter.

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Chemical compatibility of CHROMAFILmaterialsThe following table lists the chemical com-patibility of our CHROMAFILmaterials.

The chemical compatibility depends onseveral parameters such as time, pres-sure, temperature, concentration.In most cases, CHROMAFILfilters willhave only short contact with a solvent.

In these cases they may be used despiteof limited compatibility. For example, a

PTFE filter with PP housing does not liber-ate any UV-detectable substances duringfiltration of 5 ml THF, although PP showsonly limited resistance towards THF.

Solvent Material

MV

CA

RC

PA

PTFE

PVDF

PET

GF

PP

Acetaldehyde

Acetic acid, 100%AcetoneAcetonitrileAmmonia, 25%BenzenenButanolCarbon tetrachlorideChloroformCyclohexaneDiethyl etherDimethylformamide

1,4DioxaneEthanolEthyl acetateEthylene glycolFormic acid, 100%Hydrochloric acid, 30%MethanolMethylene chlorideNitric acid, 65%Oxalic acid, 10% aq.Petroleum etherPhosphoric acid, 80%

Potassium hydroxide, 1 mol/l2PropanolSodium hydroxide, 1 mol/lTetrahydrofuranTolueneTrichloroethyleneUreaWaterXylene

(Data not guaranteed) resistant, limited resistance, not resistant

PP = polypropylene, MV = cellulose mixed esters, CA = cellulose acetate,RC = regenerated cellulose, PA = polyamide, PTFE = polytetrafluoroethylene (Teflon),PVDF = polyvinylidene difluoride, PET = polyester, GF = glass fibre

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Main steps of the SPE pro-cedure1. Conditioning of the adsorbent

Conditioning of the adsorbent is nec-essary in order to ensure reproducibleinteraction with the analyte. Condi-tioning, also called solvation, resultsin a wetting of the adsorbent andthus produces an environment, whichis suitable for adsorption of the ana-lyte. Nonpolar adsorbents are usuallyconditioned with 2 3 column vol-

umes of a solvent, which is misciblewith water (MeOH, THF, isopropanoletc.), followed by the solvent in whichthe analyte is dissolved (pure matrix).Polar adsorbents are conditioned withnonpolar solvents.

After the conditioning step theadsorbent bed must not run dry,because otherwise solvation is de-

stroyed.2. Sample application (adsorption)Sample application can be performedwith positive or negative pressure witha flow rate of ~3 ml/min.

3. Washing of the adsorbent

Washing of the adsorbent is usuallyachieved with a special wash solution;however, in some cases it may not benecessary. If the polarity difference be-tween wash solution and eluent is verylarge, or if both are not miscible, dry-ing of the adsorbent bed after washingis recommended.

4. Elution

Elution with a suitable eluent shouldnot be too fast. The elution speeddepends on the column or cartridgedimension and the quantity of adsorb-ent (about 1 ml/min).

Sample pretreatmentFor direct extraction with adsorbentsthe sample matrix (sample environ-

ment) has to fulfil three conditions: The matrix has to be liquid, if pos-sible with low viscosity.

Solids should be removed from theliquid matrix.

The matrix (sample environment)should be suitable for retention ofthe analyte.

For solid samples there are differentmethods to convert the sample into asuitable matrix:

dissolution of the solid sample in asuitable solvent

lyophilisation of the sample anddissolution in a suitable solvent

extraction of the solid sample witha suitable solvent

homogenisation of the sample in asuitable solvent

In order to find the suitable solvent,one has to consider all desired samplecomponents. Also, the suitable solventshould enhance retention of the ana-lyte. For example, samples with largecontents of solids are often homog-enised in nonpolar solvents like hex-ane, while for samples with high water

content dissolution in acids, bases,buffers or very polar solvents such asmethanol is recommended.

Additionally, SPE allows to alter theproperties of the sample matrix. If,for example, natural products areextracted with methanol or acetone,the polarity of the extracts can be in-creased by dilution with water, in order

to enhance nonpolar solid phase ex-traction on the C18 material.

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Sample pretreatment for some diffi cult matrices

Matrix Problem Sample pretreatment

soil, sludgeadsorption of theanalyte on the solidmatrix

extraction with nonpolar solvents (e.g. hex-ane) and separation of interfering compo-nents on polar adsorbents

crude oil products very nonpolar matrixextraction with or dissolution in nonpolarsolvents (e.g. hexane) and separation ofinterfering components on polar adsorbents

wine, lemonadeetc.

carbohydrate-con-taining polar matrix

dilution with water and enrichment onnonpolar adsorbents; for enrichment on ionexchangers pH adjustment with buffers

ointments andcreams

differentiation foroil and water basedproducts

oil based: dissolution in nonpolar solvents(e.g. hexane) and separation of interferingcomponents on polar adsorbentswater based: dissolution in polar solvents(e.g. methanol, acetone) with subsequentdilution with water, if required; enrichment ofthe analytes on nonpolar adsorbents

oils, fats, vegetable

oils

nonpolar matrixdissolution in nonpolar solvents (e.g. hexane,petroleum ether) and separation of interfer-

ing components on polar adsorbents

cereals possible fat content

extraction with nonpolar solvents (e.g.hexane) and separation of interferingcompounds on polar adsorbents or defatsample with nonpolar solvents and extractwith polar solvents (e.g. methanol, acetone),if necessary subsequent dilution with waterand enrichment of the analyte on nonpolaradsorbents

fruit, vegetable heterogeneousmatrix, dyesextraction with polar solvents (e.g. methanol,acetone), if necessary subsequent dilutionwith water and enrichment of the analyte onnonpolar adsorbents

physiologicalsamples (serum,plasma, blood,urine)

protein contentprecipitate proteins and dilute sample withits own volume of water or suitable buffer;enrichment on nonpolar adsorbents

water humic substancesremoval of humic substances on NH2modi-fied silica and enrichment of the analyte onnonpolar adsorbents

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Standard protocols for CHROMABONDRP phases

CHROMABONDThe Simple Way

Standard SPE procedure forCHROMABONDC18 ec6 ml, 500 mg Cartridges, Cat. No. 730014

Prepare Sample

Condition6 ml MeOH

Equilibrate6 ml water

Load Samplemax. 1000 ml

Wash Impurities3 ml MeOH water (5:95, v/v)

Elute Analyte2 x 2 ml MeOH or acetone


Standard SPE procedure forCHROMABONDEasy3 ml, 200 mg Cartridges, Cat. No. 730754

Prepare Sample

Condition3 ml MeOH




Elute Analyte2 x 2 ml MeOH acetone


Standard SPE procedure forCHROMABONDHR-P3 ml, 200 mg Cartridges, Cat. No. 730108

Prepare Sample

Condition3 ml MeOH




Elute Analyte2 x 2 ml MeOH acetone (1:1, v/v)

or CH2Cl2


Standard SPE procedure forCHROMABONDPS-RP3 ml, 200 mg Cartridges, Cat. No. 730765

Prepare Sample

Condition3 ml MeOH




Elute Analyte2 x 2 ml MeOH acetone (1:1, v/v)

or CH2Cl2

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Standard protocols for CHROMABONDion exchangers


Standard SPE procedure forCHROMABONDPS-OH

3 ml, 200 mg Cartridges, Cat. No. 730396

Prepare Sample(pH 7 11)

Condition3 ml MeOH



Wash 13 ml 5% NH3in water

Wash 2 (Elute 1)*3 ml MeOH water (20:80, v/v)

Elute 22 x 2 ml MeOH acetone (1:1, v/v) + 5% AcOH

or2 x 2 ml MeOH acetone (1:1, v/v) + 20% NH3

* In this fraction neutral and basic compounds can be found


Standard SPE procedure forCHROMABONDPS-H+

3 ml, 200 mg Cartridges, Cat. No. 730690


Condition3 ml MeOH



Wash 13 ml AcOH water (5:95, v/v)

Wash 2 (Elute 1)*3 ml MeOH water (20:80, v/v)

Elute 22 x 2 ml MeOH acetone (1:1, v/v) + 5% NH3

or2 x 2 ml MeOH acetone (1:1, v/v) + 20% AcOH

* In this fraction neutral and acidic compounds can be found


Standard SPE procedure forCHROMABONDMix3 ml, 200 mg Cartridges


Condition3 ml MeOH



Wash3 ml MeOH water (5:95, v/v)

Com-pounds:

Elute 13 ml MeOH water (1:1, v/v) + 10% NH3

polar andbasic

Elute 13 ml MeOH water (1:1, v/v) + 10% AcOH

polar andacidic

Elute 13 ml MeOH acetone (1:5, v/v)

nonpolarand neutral

All solvent volumes are based on theamount of adsorbent mentioned (200or 500 mg) and have to be adjustedaccording to the size of the cartridgeused. The protocols are suggestionsfor standard procedures and shouldbe optimised (solvents, volumes,amount of adsorbent etc.) for thedemands of the investigated sampleor compound and for the subsequentanalysis (GC, HPLC etc.).

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Handling of CHROMABOND columnsand CHROMAFIXcartridges

For elution either apply pressure at thetop of the column or apply vacuum atthe column end. For this purpose sev-eral procedures are possible as shownin the figures. The adaptor shown infig. a) can be used for coupling sev-eral CHROMABOND columns of thesame or different sizes.

a) Use with a disposable syringe

With the aid of a disposable syringeand an adaptor the eluent can bepressed through the CHROMABONDcolumn.

With the aid of a syringe the requiredsolvents for conditioning, washingand elution as well as the sample it-self can be easily pushed through theadsorbent of the CHROMAFIX car-tridges without high pressures.

b) Use in a centrifuge

The same result can be obtained byusing the column in a centrifugetube.

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c) Application undervacuum

For drawing the elu-

ent through a columnor cartridge it can beplaced on an aspira-tor bottle by means ofa syringe needle, orit can be used on thevacuum manifold de-scribed below.

CHROMABONDcol-

umns and CHROMA-FIXcartridges can beused with all vacuumsystems with Luer fit-ting.

Handling of large sample volumes

For larger sample volumes MN has de-veloped the CHROMABOND LV col-umns, which are available with three

different adsorbent weights (100, 200and 500 mg) and feature a funnel-shaped reservoir of 15 ml volume.

If very large sample volumes areto be extracted, we recommendthe CHROMABOND tubing adap-tors, which consist of an adaptor forCHROMABOND columns and 1 mcoloured Teflon tubing with weight.

The package contains 4 adaptors withtubes of different colours.

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CHROMABOND vacuum manifolds for simultaneouspreparation of up to 12, 16 or 24 samples

If several samples are to be treatedsimultaneously, we recommend ourvacuum manifolds.

We supply such manifolds for up to 12,16 or 24 CHROMABOND columns orCHROMAFIX cartridges, respectively.The manifolds consist of rectangular

glass cabinets (1) with vacuum gauge(3) and a polypropylene lid (2), whichcan hold the columns or cartridges.The replaceable valves (4) on the lidallow individual vacuum control foreach solid phase extraction column, ifrequired.

The cabinet is fitted with a variablerack (5) with exchangeable partitions,which accept a wide variety of vesselslike test tubes, measuring flasks, scin-tillation vials, autosampler vials, plasticvials and many more. With the controlvalve (6) the vacuum in the chambercan be adjusted and read from thegauge.

There are several possibilities for ap-plying different sample volumes. Smallsamples can be applied directly to theCHROMABOND column. For mediumsize samples we have developed ourCHROMABOND LV columns (7) with15 ml sample reservoir. Especially forthis column type we offer a vacuummanifold with 16 positions, becausewith the manifold for 24 columns only

every second position can be used.Alternatively, you may use the poly-propylene sample reservoirs (30 or 70ml) from our programme of SPE ac-cessories, which can be fitted onto theCHROMABOND column with the aidof an adaptor (8). Sample reservoirs fitdirectly onto the upper Luer fitting ofthe CHROMAFIXcartridges. For largesample volumes we recommend ourCHROMABOND tubing adaptors (9),which fit onto the CHROMABONDcol-umns. The other end of the tubing isplaced into the sample, which, by ap-plying vacuum, is continuously drawninto the CHROMABONDcolumn.

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For special applications, which requiremaximum protection from cross con-tamination we supply chrome-plated

brass valves and stainless steel con-nectors, the application of which isshown in the figure below. These spe-cial stainless steel needles are fittedthrough the lid; thus the sample onlyhas contact with the inert needle andcan flow directly into the receptacle.

Standard configuration (left) comparedto the cross-contamination-free elu-tion with stainless steel or PTFE con-nectors (right)

If the eluate has to be evaporated, thiscan be performed with the so-calleddrying attachment (10, see figure be-

low). This special lid has a gas connec-tor on one side (11), from which thegas is fed simultaneously to the 12 or24 stations (12). Thus 12 or 24 elu-ates can be evaporated simultaneouslyby just changing the lid and applying astream of inert gas, e.g. nitrogen.

For collection of the sample, vials andvial accessories are available on re-quest. Please ask for further informa-tion.

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CHROMABONDPP tank for vacuum manifoldfor 12 columnsThis polypropylene tank is a supple-ment for the 12-column vacuum man-ifold and allows to collect solvent resi-dues (matrix, washing solutions) whichare to be discarded, avoiding any con-tamination of the vacuum manifold it-self. The CHROMABONDPP tank canbe used as follows:

If you want to collect the solvent resi-dues (matrix residues) during solid

phase extraction, place the PP tankinto the vacuum manifold as shown inthe following figure.

The lid is placed onto the glass cabinet(see next figure), and the SPE columns orcartridges are placed onto the valves.

Now the SPE procedure can be started.The sample matrix, which has passedthrough the adsorbent and is to bediscarded, is collected in the PP tank. Ifthe procedure requires washing of theCHROMABONDcolumns or CHROMA-FIX cartridges after enrichment, thewashing solutions can also be collect-ed in the PP tank.

Before eluting the analyte(s), the lid is

taken off and the PP tank is removedfrom the vacuum manifold. The tankhas two small handles on the sides forconvenient removal.

After the PP tank has been removed,the elution can be performed as usu-al by placing the rack with the eluateflasks into the glass cabinet. When thelid is put back onto the chamber, care

has to be taken that the needles on thelower side of the lid are inside the re-spective flasks.

Solvent residues should be thoroughlyremoved from the PP tank after use.You may rinse the tank and use itagain. Application of the PP tank cansave valuable time and is very conven-ient, because it makes cleaning of the

vacuum manifold much easier.

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Handling of CHROMABONDMULTI 96

The CHROMABOND MULTI 96 areparticlularly designed for the use inall common robotic workstations orcommercially available liquid handlingsystems. Alternatively, the use of mul-ti-channel pipettors facilitates a man-ual liquid transfer. The extraction iscarried our using the CHROMABONDMULTI 96 vacuum manifold. With thehelp of the control valve the vacuum ofthe manifold can be adjusted leading

to an optimum flow rate through theCHROMABONDMULTI 96 SPE plate.

A reservoir tank and 96-well collectionplates (96 x 0.5 or 96 x 2 ml) madeof polypropylene can be supplied asaccessories. An interesting alternativefor collection of the eluates is a col-lection rack, which can be fitted withtwelve 8-well strips of polypropylene

tubes (each 1 ml). If you have to workon less than 96 samples, you can sealindividual rows of the 96-well platewith a PTFE-covered rubber pad.

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Biological samplesand natural

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Alkaloids: nicotine and cotinine from plasmaMN Appl. No. 300070

Column type:

CHROMABONDC18 ec / 1 ml / 100 mgCat. No. 730011

Sample pretreatment: mix 1 ml plasma with 1 ml saturated ammonium chloride solution andadjust to pH 10 with aqueous ammonia solutionColumn conditioning: 2 column volumes methanol, then 2 column volumes dist. waterSample application: slowly force or aspirate the sample through the columnColumn washing: 2 x 200 l saturated ammonium chloride solution acetonitrile (9:1, v/v); dry

column for 5 min under vacuumElution: 2 x 250 l dichloromethane

Alkaloids from plant extractsMN Appl. No. 300080

H. Wiedenfeld, R. Lebada and B. Kopp,

Dtsch. Apothekerzeitung 135(1995) 17 26Compounds investigated:pyrrolizidine alkaloids

from coltsfoot extracts (senecionine, senkirkin) Column type:CHROMABONDDiol / 3 ml / 500 mgCat. No. 730053

Sample pretreatment:Commercial tea powder:

Dissolve 16.6 g tea powder in 1.5 l boiling water (= 10 cups of tea). Adjust pH to 2.5 3with citric acid and evaporate to 500 ml. Wash aqueous phase several times with ether andCH2Cl2. Make ammoniacal and extract with CH2Cl2.

Commercial drug (dried coltsfoot leaves, 15 g sample each):Different extraction methods were compared: alcoholic Soxhlet extraction of a powered drug sample cold maceration of coarsely cut drug with 1.5 l water during 30 min under stirring BGA procedure (German health administration): add 1.5 l boiling water to the coarsely cut

drug sample and let stand 15 min AB procedure (Austrian pharmacopoeia): wet coarsely cut drug sample with water in a

mortar and let stand 5 min; then add 1.5 l boiling water and let stand 30 minAll extracts were processed as described for the tea powder above.Column conditioning: 3 ml dichloromethaneSample application: apply dichloromethane extract from the sample pretreatment stepColumn washing: 3 ml dichloromethaneElution: methanol acetonitrile (1:1, v/v)

For further analysis we recommend GC on an OPTIMA5 column.

Alkaloids


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Pyrrolizidine alkaloids and their N-oxides in plant materialMN Appl. No. 302330

G. Hsch et al. Phytochemical Analysis 7(1996) 284 288

Column type:CHROMABONDC18 / 3 ml / 500 mgCat. No. 730003

Sample pretreatment: approximately 2.5 g plant material are extracted with methanol in a soxhletapparatus for 6, 16 and 21 h, respectively. After evaporation to dryness the residue is dissolved in100 ml methanol. An aliquot (8.0 ml) is evaporated to dryness and suspended in 2.5 % HCl (3 ml).This suspension was filtered and the filter was washed with 2.5 % HCl (1 ml).Column conditioning: 3 ml methanolSample application: slowly force or aspirate the acidic solution from the sample pretreatmentthrough the column

Column washing: 3 ml of waterElution:8 ml of 25 % methanol; evaporate to dryness under reduced pressure and dissolve in1.0 ml methanol

Further analysis: HPLCRecovery rates:The total recovery of alkaloids decreased with the time of soxhlet extraction.While a PA yield of 0.55 % was determined in the plant material after a 6 h extraction, this droppedto 0.49 % after 16 h and even to 0.44 % after a 21 h extraction. This finding was confirmed usingthe pure N-oxides in the absence of plant material. Recovery of the N-oxides dropped from 99.5to 84.1 % within 24 h of soxhlet extraction.

Isolation of scopolamine from tobacco rootsMN Appl. No. 303410

A. Maslak, Dissertation (2002), Mathematisch-

Naturwissenschaftlich-Technische Fakultt derMartin-Luther Universitt Halle-Wittenberg, Germany


Sample pretreatment: 5 g ground tobacco roots from 8 weeks old plantsof Nicotiana tabacumcv. SamsunNN were extracted with 15 ml of 50 %

MeOH for 30 min and filtered with a 100 m nylon filter and a cellulosemembrane (45 m). The filtered sample was diluted with water to 10 %MeOH.Column conditioning: 1 column volume methanol, then 1 column volume waterSample application: slowly force or aspirate the sample extract through the columnColumn washing: three times with methanol water (1:9, v/v)Elution:20 ml methanol; evaporate the combined eluates to 1 ml in a vacuum centrifuge

Further analysis: HPLC

Biological samples and natural compounds


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Aromatic amines from urine and serumMN Appl. No. 300090

Column type:


Sample pretreatment: adjust sample to pH 10 with 5 M sodium hydroxide solutionColumn conditioning: 1 ml methanol, then 1 ml dist. waterSample application: slowly force or aspirate 500 l sample through the columnColumn washing: 1 ml dist. water; then dry the column with air for 5 minElution: 200 l ethyl acetate; concentrate eluate in a stream of nitrogen

1,2,3,4-tetrahydroisoquinolines and other potentially neurotoxic compounds

MN Appl. No. 303480P. Pagel et al., J. Chromatography B, 746(2000) 283 295Compounds investigated:model compounds for method development (see table below)

Column type:

CHROMABONDC18 ec / 30 mg(self-packed by the author, available on request)

Column conditioning: 1 ml methanol, then 1 ml waterSample application: force or aspirate 500 l plasma sampleor reference solution through the columnElution:

Fraction F-1:effluent after load of 500 l water

Fraction F-2:effluent after load of additional 500 l water andapplication of pressure to remove water completely,subsequently addition of 500 l methanol to dry the columnFraction F-3:effluent after load of additional 500 l methanolFraction F-4:effluent after load of additional 500 l methanol

Further analysis: HPLC with UV (215 and 240 nm) and

fluorescence detection (ex285, em315/340 nm)Recovery rates:(in the presence of 1-hexanesulfonic acid sodium salt)

Compound Recovery [%]

F-1 F-2 F-3 F-4 R1 R2DOPA*


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Catecholamine metabolites from urineMN Appl. No. 300120

Compounds investigated:vanillylmandelic acid (R = CH(OH)-COOH) and

homovanillic acid (R = COOH)

Column type:CHROMABONDSB (SAX) / 3 ml / 500 mgCat. No. 730079

Sample pretreatment: collect 24 h urine (preserved with 0.1 M hydrochloric acid) and store at 4C or 20 C, resp. Prior to extraction dilute sample 1:1 with water and adjust the pH value to 7.5with 0.5 M sodium hydroxide solution.

Column conditioning: 2 column volumes methanol, then 2 column volumes dist. waterSample application: slowly force or aspirate 1 ml sample through the columnColumn washing: 2 x 2.5 ml dist. waterElution: 2 x 2.5 ml 1.5 M sodium hydroxide solution

Catecholamine metabolites from plasmaMN Appl. No. 300100

Compounds investigated:homovanillic acidColumn type:

CHROMABONDC8 / 3 ml / 500 mg andCHROMABONDSB (SAX) / 1 ml / 100 mgCat. Nos. 730023 and 730078

Sample pretreatment: mix 1 ml plasma sample with 50 l 0.1 M EDTA (if desired, add 30 l iso-homovanillic acid in 0.01 M hydrochloric acid as internal standard) and add 200 l 1 M hydrochlo-ric acid. Dilute with 2 ml water.Column conditioning:C8: 5 ml methanol, then 5 ml dist. waterSB: 1 ml methanol, then 5 ml 1 M sodium acetate buffer pH 6.0, finally 1 ml dist. waterSample application: slowly force or aspirate the plasma sample through the C8 columnColumn washing: 2 ml waterElution of C8 column: 2 ml methanol water (50:50, v/v); dilute eluate with 1 ml 0.1 M sodiumacetate buffer pH 6Sample application: force or aspirate the diluted eluate from the C8 column through the SBcolumn with 0.5 ml/minElution of SB column:2 x 300 l 1 M hydrochloric acid with 0.5 ml/min



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Catecholamines from urineMN Appl. No. 300110

Column type:

CHROMABONDSA (SCX) / 3 ml / 500 mgCat. No. 730077

Sample pretreatment: mix 1 3 ml urine with 2 3 drops of a 1 M sodium carbonate solutionColumn conditioning: 2 column volumes methanol, then 2 column volumes dist. waterSample application: force or aspirate pretreated sample through the columnColumn washing: 2 column volumes dist. waterElution: 3 x 500 l 0.5 M hydrochloric acid; then adjust the eluate to pH 3 with 1 M sodium car-bonate solution

Xanthines: caffeine and theophylline from serumMN Appl. No. 300680

Column type:


Sample pretreatment: mix 1 ml serum with about 1 ml 0.01 M Tris buffer pH 7Column conditioning: 2 column volumes methanol, then 2 column volumes0.01 M Tris buffer pH 7Sample application: slowly force or aspirate the sample through the columnColumn washing: 2 column volumes dist. water

Elution: 2 x 300 l methanol

Xanthines: theophylline from serumMN Appl. No. 300690

Column type:


Sample pretreatment: mix 100 l serum with 100 l internal standard (20 g/ml -hydroxyethyl-theophylline in 0.1 M phosphate buffer pH 4.0)Column conditioning: 2 column volumes methanol, then 2 column volumes dist. waterSample application: slowly force or aspirate the sample through the columnColumn washing: 2 column volumes dist. waterElution: 2 x 200 l methanol

Catecholamines Xanthines


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Benzalkonium chloride from plasmaMN Appl. No. 301950

Column type:


Sample pretreatment: dilute 2 ml plasma with 4 ml waterColumn conditioning: 1 column volume methanol, then 1 column volume waterSample application: slowly force or aspirate the diluted sample through the columnColumn washing: 3 x 3 ml water, then 2 x 3 ml methanol, finally 2 x 3 ml ethyl acetateElution: 4 ml methanol ethyl acetate (1:1, v/v) containing 0.01 % ammonium chloride; concen-trate eluate in a stream of nitrogen

Removal of chlorophyll from plant cellsMN Appl. No. 300010

Column type:CHROMABONDSA (SCX) / 3 ml / 500 mgCat. No. 730077

Sample pretreatment: homogenise plant cells 2 min with 100 ml dichloromethane acetone(3:1, v/v)Column conditioning: place 0.5 g dry Na2SO4onto the SA packing and condition column with1 column volume dichloromethane acetone (3:1, v/v) (special columns containing 500 mgCHROMABONDSA and 500 mg Na2SO4are available on request)Sample application: slowly force or aspirate 4 ml sample through the column and collect thechlorophyll-free eluate. When chlorophyll is eluted, reduce the sample volume.1stelution: 0.5 ml dichloromethane acetone (3:1, v/v), combine the eluate with the chlorophyll-free eluate2ndelution: if the substance to be determined still remains on the column together with the chlo-rophyll, elute the desired compounds with a more polar eluent (acetone, acetonitrile, methanol) oran eluent of lower pH value

Porphyrins: protoporphyrin from fecesMN Appl. No. 300500

Column type:


Sample pretreatment: mix 200 250 mg feces with 10 ml 0.5 g/lTween 40 solution and shake 30 s or sonicate. Mix 0.5 ml of thissolution with 2 ml 3.125 M oxalic acid 0.125 M iron sulphatesolution 0.5 g/l Tween 40 solution and shake thoroughlyColumn conditioning: 3 ml 0.05 mol/l HCl, then 2 ml methanol glacial acetic acid water (75:2:23, v/v/v)Sample application: slowly force or aspirate 250 l of the pretreated sample through the columnColumn washing: 3 ml MeOH HAc water (75:2:23, v/v/v)Elution: 5 ml methanol 30 % phosphoric acid (3:1, v/v)



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Bacterial cleavage of nitrogen to sulfone bonds in sulfamide and 1H-2,1,3-benzo-thiadiazin-4(3H)-one 2,2-dioxide: formation of 2-nitrobenzamide by Gordoniasp.

MN Appl. No. 302530

U. Rein, A. M. Cook, FEMS Microbiology Letters 172(1999) 107 113Compounds investigated:sulfamate, sulfamide, 1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide,nitrobenzamide-2

Column type: Benzothiadiazinone dioxide

CHROMABONDHR-P / 3 ml / 200 mgCat. No. 730108

Sample pretreatment: adjust sample to pH 9 with 10 mol/l NaOHColumn conditioning: 2 ml methanol, 2 ml acetonitrile and 2 ml NaOH (c = 105mol/l)Sample application: force or aspirate the sample through the column with 10 ml/minColumn washing: 2 ml dist. water; dry cartridge under vacuum for 5 minElution:3 x 1 ml methanol acetonitrile (1:1, v/v)

Further analysis: reversed-phase HPLC using a column NUCLEOSIL100-5 C18with gradientelution

Cyclic peptides microcystin and nodularin from algal cells and waterMN Appl. No. 302631 / 302632

C. Hummert, et al. Chromatographia 50(1999) 173 180 Column type:

CHROMABONDC18 ec / 3 ml / 500 mg

Cat. No. 730013Algae samples:

Sample pretreatment: algae samples are filtered through a 0.45 m glass fiber filter. The filterscovered with algae cells and 1.0 ml of a mixture of water methanol (50:50, v/v) are sonicated for20 min and finally centrifuged. The supernatant is filtered through a nylon filter with 0.45 m poresize (Cat. No. 729011).Column conditioning: 5 ml methanol, 5 ml water (0.05 % TFA)Sample application: slowly force or aspirate 1 ml of algae extract through the columnWater samples:

Column conditioning: 2 ml methanol, 5 ml water (0.05 % TFA)

Sample application: slowly force or aspirate 500 ml water sample through the columnFor all samples:

Column washing: 10 ml water containing 0.05 % TFA, then 5 ml water containing 0.05 % TFA methanol (80:20, v/v)Elution: 5 ml methanol; evaporate to dryness using rotary evaporation for the main volume and astream of nitrogen for the last 200 l. Redissolve in 200 l methanol water (50:50, v/v).


Miscellaneous


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Cyanopeptolins from cyanobacteriaMN Appl. No. 300190

C. Martin et al., J. Antibiotics 46(1993), 1550 1556Compounds investigated: cyanopeptolines A, B, C and D

A: R = NH-C(NH)-NH2

B: R = CH2-NH2

C: R = CH2-NH-CH3

D: R = CH2-N(CH3)2


Sample pretreatment: lyophilised cells of cyanobacterium microcystissp. PCC 7806 (25 g drysubstance) are extracted with 500 ml MeOH for 2 h at room temperature. After centrifugation theclear supernatant is concentrated to 50 ml using a rotation evaporator and then filled to 500 mlwith dist. water.Column conditioning: 2 column volumes methanol, then 2 column volumes dist. waterSample application: slowly force or aspirate the prepared sample through the column

Column washing: 10 ml dist. waterElution: force or aspirate 30 ml methanol through the column; concentrate to 3 ml

Pyridinium crosslinks from urineMN Appl. No. 302070

Compounds investigated:pyridinoline, deoxypyridinoline Column type:

CHROMABONDCrosslinks, 3 ml, 300 mg,Cat. No. 730458

Sample pretreatment: 250 l urine and 50 l of an internal standard (e. g. pyridoxine) are hy-drolised in 250 l conc. HCl at about 100 105 C for 12 16 h. Then 2.5 ml wash solution (n-bu-tanol glacial acetic acid 80:20, v/v) are added to the hydrolysate.Column conditioning: 5 ml of the wash solutionSample application: force or aspirate the pretreated sample through the column. Discard the flow-through.Column washing: 15 25 ml of the wash solution.Elution:force or aspirate 3 5 ml dist. water through the column



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Diaminopyridine from urineMN Appl. No. 300220

Column type:


Column conditioning: 1 column volume methanol, then 1 column volume dist. waterSample application: slowly force or aspirate 1 ml sample through the columnColumn washing: 2 ml dist. water, then 2 x 250 l acetonitrileElution: 100 l of a solution of 100 mg trimethylammonium chloride in acetonitrile water (80:20,v/v), then 100 l dist. water

Amino acids from urine

MN Appl. No. 300230 Column type:

CHROMABONDSA (SCX) / 1 ml / 100 mgCat. No. 730076

Sample pretreatment: mix 50 l urine with 450 l water and adjust to pH 1.1 with about 5 l conc.hydrochloric acidColumn conditioning: 5 ml methanol, then 5 ml methanol 0.1 M hydrochloric acid (1:1, v/v),finally 5 ml 0.1 M hydrochloric acidSample application: slowly force or aspirate the sample through the columnColumn washing: 700 l water

Elution:900 l 1 M aqueous ammonia solution; then freeze dry, if you do not analyse your sampleimmediately

Enrichment of viscotoxines from Viscum AlbumMN Appl. No. 303040

Private communication: S. Jger, ABNOBA Heilmittel GmbH, Pforzheim, Germany Column type:

CHROMABONDPCA / 6 ml / 500 mgCat. No. 730483

Sample pretreatment: 2 to 6 ml of Viscum Albumextracts are diluted in 5 to 15 ml waterColumn conditioning: 2 ml methanol, 2 x 2 ml 0.02 M ammonium acetate bufferSample application: slowly force or aspirate 2 to 6 ml sample through the columnColumn washing: 2 x 2 ml 0.02 M ammonium acetate bufferElution:10 ml 4 M acetic acid buffer


Miscellaneous


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Bile acids from serumMN Appl. No. 300250

Column type:


Sample pretreatment: mix 1 ml serum with 4 ml 0.1 M sodium hydroxide solution and heat 15 minto 64 CColumn conditioning: 2 column volumes methanol, then 2 column volumes dist. waterSample application: slowly force or aspirate the sample through the columnColumn washing: 1 column volume dist. waterElution: 2 x 1 ml methanol

Flavonols and flavones from parsley cell suspensionsMN Appl. No. 302260

J. Hempel et al. Nahrung 43(1999) 201 204 Column type:

CHROMABONDC18 / 3 ml / 500 mgCat. No. 730003

Sample pretreatment: samples of 4.44 g lyophilised cell culture are dispersed in 200 ml methanol water (70:30, v/v) and mixed for 15 min with an Ultra-Turrax homogeniser. The extract solution isremoved by suction on a G3 glass filter covered with a paper filter. The filtrate is evaporated at 35C to remove the methanol.Column conditioning: 3 ml of methanol and 5 ml of waterSample application: slowly force or aspirate the sample through the columnColumn washing: with waterElution:5 ml of methanol, evaporate to dryness and suspend in 1 ml of water

Further analysis: HPLC, LC/MS

Flavonoids from tomato peelMN Appl. No. 300150

Column type:

CHROMABOND

PA / 6 ml / 500 mgCat. No. 730007Sample pretreatment: extract 0.5 g dried tomato peel 1 h at 70 C with 30 ml methanol water(70:30, v/v). Then filter and concentrate to 4 5 ml under vacuum.Sample application: slowly force or aspirate the sample through the columnColumn washing: 8 ml dist. waterElution: 6 ml methanol



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Analysis of flavonols of Sedum telephiumL. leaves by capillary electrophoresisand HPLC/MS

MN Appl. No. 302570

S. Sturm et al. Chromatographia 50(1999) 438 433 Column type:

CHROMABOND

C18 / 100 mg / 1mlCat. No. 730001Method A for development of CE and HPLC/MS methods:Sample pretreatment: dissolve 10 mg lyophilised juice from S. telephiumleaves in 0.1 ml offormic acid (0.1 mol/l)Sample application: slowly force or aspirate the sample through the columnElution:with water containing increasing amounts of MeOH. Flavonol glycosides eluted with 50 %MeOH are evaporated to dryness. For analysis the residue is dissolved in 1 ml of a mixture ofMeOH, acetone and water (50:25:25, v/v/v).Method B for quantitative determination:Sample pretreatment: 2 g of the fresh plant material are frozen with liquid nitrogen, lyophilised

and ground. After addition of 1.70 mg of rutin, 500 mg is extracted with a mixture of MeOH,acetone and water (50:25:25, v/v/v) using an Ultra-Turrax T25, centrifuged and filtered. This pro-cedure is repeated 7 times and the solvent of the collected filtrates is evaporated. The residue isdissolved in 5.00 ml formic acid (50 mM).Sample application: slowly force or aspirate 0.50 ml of the solution through the columnElution: with water, 10 %, 25 % and 50 % MeOH (2 ml each). The last fraction containing the flavo-nol glycosides was evaporated and dissolved in 1.00 ml of a mixture of MeOH, acetone and water(50:25:25, v/v/v). A duplicate was prepared in the same way.

Further analysis: LC/MS

Isoflavones from plant tissuesMN Appl. No. 300140

C

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