Patent Document:

for the preparation of the column packing material the redispersable , non - porous , monosized silicon dioxide microspheres must be first redispersed in a non - protic solvent , such as acetonitrile , propionitrile , under the action of ultrasound . after redispersion the silylating agent is added , the suspension heated to reflux . after isolation and drying at 120 ° c ./ 10 - 3 torr the powder is treated again with the same silylating agent in order to complete the surface layer . another possibility is to wet the slightly agglomerated , redispersable powder by a non - polar type solvent such as cyclohexane , toluene or xylene , add the silylating agent ane treat the stirred suspension by ultrasound where the product is partly silylated and forms a monosized suspension . after heating to reflux for 10 - 30 h the product is isolated in a centrifuge . elemental analysis shows that a nearly compact layer of alkyldimethylsiloxy layer is formed . the product may be treated a second time with the same silylating agent in order to complete silylation . using the derivatized final products short chromatographic columns ( 33 × 4 . 6 mm ) were packed . in the following the preparation of the adsorbents and their stability tests are described . furthermore , the separation of low molecular weight compounds as well as that of biomolecules are illustrated in a few typical examples . the time of re - equblibration of the column , ready to start a new analysis after gradient elution is typically 1 - 2 min i . e . is reduced by a factor 5 - 10 . eluents are environmental friendly i . e . they contain much less organic component . preparation of kovasil ms - dmb . the starting material was kovasil ms from chemie uetikon ( uetikon , switzerland ) composed of redispersable , non - porous , monosized silicon dioxide microspheres , rehydrated in water for 20 h . specifications : a quantity of 10 . 0 g of the product was redispersed in 200 ml acetonitrile applying ultrasonic irradiation during 15 min . ( 3 , 3 - dimethylbutyl ) dimethyl ( dimethylamino ) silane ( 67 . 5 mg ) was added to the suspension which was refluxed under an argon atmosphere during 12 h . the partly silylated product was isolated by centrifugation and washed with cyclohexane and finally dried . the dried product was transferred into a glass ampoule , the same silylating agent ( 60 mg ) was added then the ampoule was cooled , evacuated and sealed . the closed ampoule was kept at 150 ° c . during 50 h . after cooling the ampoule was opened and the white powder was washed with cyclohexane and dried . surface concentration of the ( 3 , 3 - dimethylbutyl ) dimethylsiloxy groups was γ sox = 4 . 0 μmol m - 2 . in the following this stationary phase will be designated as kovasil ms - dmb . ( the stationary phase silylated with the same method but with use of tetradecyl - dimethyl ( dimethyilamino ) silane as silylating agent will be designated as kovasil ms - c14 ). test of a column packed with kovasil ms - c14 . separation of a test mixture composed of toluene ( 1 ), butylbenzene ( 2 ) and pentylbenzene ( 3 ). experimental : column : 33 × 4 . 6 mm ; elution mode : isocratic ; mobile phase : acetonitrile / water ( an / w = 50 / 50 by volume ); flow rate : 0 . 9 ml min - 1 ; pressure : 20 mpa ; temperature : ambient ; detector : uv 254 nm ( see fig1 ). theoretical plate numbers , n x : n toluene : 4100 ; n butylbenzene : 6150 ; n pentylbenzene : 7980 . the increase of number of theoretical plates as a function of retention time was explained by extracolumn contributions of the chromatographic system to peak - broadening ( with longer retention this contribution has a smaller effect ). hydrolytic stability of the stationary phases , kovasil ms - c14 and kovasil ms - dmb . experimental : columns : 33 × 4 . 6 mm ; mobile phase is a mixture of acetonitrile / water ( an / w = 50 / 50 by volume ) containing 0 . 12 % trifluoroacetic acid ( tfa ); temperature : 80 ° c . ; flow rate : 1 . 5 ml min - 1 ; pressure : 16 mpa . the columns were connected periodically to the chromatographic system ( every 20 h ) and the mixture of example 2 was injected . relative retention as a function of time are shown in fig2 . during the whole test period ( 400 h ) the number of theoretical plates remained constant . separation of a protein mixture of analytical importance on kovasil ms - dmb components : 1 ribonuclease , 2 cytochrom c ( horse ), 3 cytochrom c ( bovin ), 4 lysozyme , 5 conalbumin , 6 myoglobin , 7 β - lactoglobulin b , 8 β - lactoglobuihn a , 9 chymotrypsinogene . experimental : column : 33 × 4 . 6 mm ; elution mode : multilinear gradient by mixing a and b where eluent a is a mixture of an / w = 20 / 80 and eluent b is an / w = 90 / 10 both containing 0 . 12 % trifluoroacetic acid ( tfa ); elution program : 0 → 0 . 5 min 3 . 6 → 27 % b , 0 . 5 → 1 min 27 → 29 % b , 1 → 1 . 2 min 29 → 37 % b , 1 . 2 → 2 min 37 → 39 % b , keep and return ; flow rate : 3 . 0 ml min - 1 ; pressure at start 35 . 1 mpa ; temperature : 80 ° c . ; detector : wv 215 nm ( see fig3 ). separation of β - lactoglobulins on kovasil ms - dmb . components : 1 β - lactoglobulin b , 2 β - lactoglobulin a . experimental : column : 33 × 4 . 6 mm ; elution mode : linear gradient by mixing a and b where eluent a is a mixture of an / w = 20 / 80 and eluent b is an / w = 90 / 10 both containing 0 . 12 % trifluoroacetic acid ( tfa ); elution program : 0 → 0 . 8 min 28 → 100 % b , keep and return ; flow rate : 3 . 6 ml min 1 ; pressure at start 35 mpa ; temperature : 90 ° c . ; detector : uv 215 nm ( see fig4 ). separation of octapeptides on kovasil ms - dmb . the seven octapeptides analysed have the following structure : tyr - ile - prox - ala - glu - lys - ile with x : 1 x = lys , 2 x = asn , 3 x = ala , 4 x = glu , 5 x = lie 6 x = phe 7 x = leu . experimental : column : 33 × 4 . 6 mm ; elution mode : multilinear gradient by mrixing a and b where eluent a is a mixture of an / w = 3 / 97 and eluent b is an / w = 90 / 10 both containing 0 . 05 % trifluoroacetic acid ( tfa ); elution program : 0 → 3 min 1 → 9 . 7 % b , 3 → 7 min 9 . 7 → 50 % b , keep and return ; flow rate : 1 . 5 ml min - 1 ; pressure at start : 22 mpa ; temperature : 40 ° c . ; detector : uv 215 nm ( see fig5 ). separation of frypsin digest of serum albumin on kovasil ms - dmb . experimental : column : 66 × 4 . 6 mm ; elution mode : multilinear gradient by mixing a and b where eluent a is a mixture of an / w = 2 / 98 and eluent b is an / w = 90 / 10 both containing 0 . 1 % trifluoroacetic acid ( tfa ); elution program : 0 → 1 . 8 min 5 → 12 % b , 1 . 8 → 3 . 8 min 12 → 32 % b , 3 . 8 → 6 min 32 → 50 % b , keep and return ; flow rate : 1 . 4 ml mim - 1 ; pressure at start : 37 mpa ; temperature : 37 ° c . ; detector : uv 215 nm ( see fig6 ). separation of proteins on kovasil ms - c14 . components : 1 insulin ( bovin ), 2 insulin ( porcine ), 3 cytochrom c ( horse ), 4 cytochrom c ( bovin ), 5 β - lactoglobulin b , 6 β - lactoglobulin a . experimental : column : 33 × 4 . 6 mm ; elution mode : linear gradient by mixing a and b where eluent a is a mixture of an / w = 5 / 95 and eluent b is an / w = 90 / 10 both containing 0 . 12 % trifluoroacetic acid ( tfa ); elution program : 0 → 3 . 5 min 7 → 45 % b , keep and return ; flow rate : 1 . 6 ml min - 1 ; pressure at start 33 mpa ; temperature : 37 ° c . ; detector : uv 215 nm ( see fig7 ). 1 . melander , w . r . and horvath , cs . ; in horvath , cs . ( ed . ), high performnnce liquid cihrontafography -- advances and perspectives , vol . 2 , academic press , new york 1980 2 . jelinek , l ., erbacher , c ., sz . kovats , e ., eilropean patent 0 574 642 a1 ( 1992 )

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