HPLC '89. Poster Presentation. Preparative Scale Separations of Oligosaccharides on Polymeric HPLC Packing Materials
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1 Essentials in " HPLC '89 Poster Presentation Preparative Scale Separations of Oligosaccharides on Polymeric HPLC Packing Materials G. Vella and K. Jones Waters Chromatography Division of Millipore S. Hirani and D. McNeilly Genzyme Corporation, Boston, MA D. Cummmg, R. Camphausen and D. Benjamin Genetics Institute, Andover, MA Water,. The absolute essentia I WatersMill,poreChromatographYcorporation Division _A/'_ ters for bioresearch. 34 Maple St. Divisionof MILLIPORE Milford, MA O1757 (508) /
2 PREPARATIVE SCALE SEPARATIONS OF OLIGOSACCHARIDES ON POLYMERIC HPLC PACKING MATERIALS C. Phoel_ 1I G. Vella 1, K. Jones 1! S. Hirani 2, D. McNeilly 2, D. Cumming 3 R. Camphausen 3, and D. Benjamin 3 1 Millipore Corporation, Waters Chromatography Division, 34 Maple Street, Milford, MA Genzyme Corporation, 75 Kneeland Street, Boston, MA Genetics Institute, One Burtt Road, Andover, MA Objective: To demonstrate multi-mode chromatographic approaches using polymeric HPLC packing materials in the preparative purification of oligosaccharides. In elucidating oligosaccharide structures, identification of an unknown structure based on chromatographic retention time alone, is not convincing because of the immense number of possible structural isomers which may ce-elute with the compound of interest. Unequivocal structure determination of oligosaccharides requires the use of high field NMR and/or FAB-mass spectrometry. One drawback of these techniques is that they require a large quantity of a homogeneous sample. High-performance liquid chromatography (HPLC) has been used to purify large quantities of oligosaccharides in high purity from complex mixtures. Described in this paper are multi-mode chromatographic approaches, using stable polymeric columns, which demonstrate the usefulness of HPLC in the isolation and purification of large quanitities of oligosaccharides. Example one demonstrates the use of the Prep Glyco-Pak TM N column for the isolation and purification of neutral complex and high mannose oligosaccharides. Example two illustrates a loading optimization study on prototype Glyco-Pak II DEAE material packed in a glass AP column for the purification of sialylated oligosaccharides from fetuin. Example three illustrates the purification of chitin oligomers using the Glyco-Pak N and Ultrahydrogel TM DP columns.
3 INTRODUCTION: In oligosaccharide structure elucidation work, no identification of an unknown structure based on chromatographic co-migration is convincing because of the immense number of possible structural isomers which might have identical retention times. Unequivocal structural determination of oligosaccharides requires the usage of high field NMR and/or FAB-mass spectrometry. One drawback of these techniques is that they require a large quantity of a homogeneous sample. Highperformance liquid chromatography (HPLC) has been used to purify large quantities of oligosaccharides in high purity from complex mixtures. Described in this paper are several examples demonstrating the usefulness of HPLC with stable polymeric columns for the isolation and purification of large quanities of homogeneous oligosaccharides. Example one demonstrates the use of the Prep Glyco-Pak TM N column for the isolation and purification of neutral complex and high mannose oligosaccharides. Example two illustrates a loading optimization study on a prototype Glyco-Pak II DEAE in a glass AP column for the purification of sialylated oligosaccharides from fetuin. Example three uses a multichromatographic mode approach (using two polymeric columns) for the purification of chitin oligomers.
4 Prep Glyco-PakTMN Purification of high mannose and complex oligosaccharides
5 Hydrazine released ovomucoid DETECTION: UV at 200 nm oligosaccharides INJECTION:: 20 mg of mixture COLUMN: Prep Glyco-Pak TM N (22 x 600 mm) ELUENT: Acetonitrile/Water (70:30) FLOWRATE: 7.25 ml/min UI o ' I ' 1 ' I ' I ' I ' x 102 minules
6 The maximum load on the analytical (7.8 x 300 mm) Glyco-PakTM N column is 1 to 2 mg. Illustrated are scale-up isolations using the Prep Glyco-Pak N column (22 x 600 mm) at a flow rate of 7.25 ml/min, for neutral complex oligosaccharides (ovomucoid, Figures 1-3) and high mannose oligosaccharides (Figure 4). Based on scale up strategies, the load on the Prep Glyco-Pak N column is approximately 30 mg. Two injections of ovomucoid neutral complex oligosaccharides at sample loads of 20 mg and 40 mg show little deterioration of the analytical separation. Figure 4 shows the baseline separation of high mannose oligosaccharides on the Prep Glyco-Pak N column utilizing a sample load of 23 mg.
7 Hydrazine released ovomucoid nc DETECTION: UV at 197 nm oligosaccharides INJECTION:: 200 pg of mixture COLUMN: Glyco-Pak TM N (7.8 x 300 mm) ELUENT: Acetonitrile/Water (70:30) FLOW RATE: 1 ml/min. I Percent ! 20! 0 rtl- - -tt-rfq-ill t]-t-11-rrrr-! i 11-rr11-r1 i i '! ri i i, I i-[-l-i-t'i i i T BO Minutes
8 Reduced Endo H released high manno_ DETECTION: UV at 200 nm oligosaccharides INJECTION: 23 mg of mixture C.,OLUMN: Prep Glyco-Pak TM N (22 x 600 mm) PEAK ID'S: 1. Man 3 GN 1 ELUENT: Acetonitrile/Water (70:30) 2. Man 4 GN 1 FLOWRATE: 7.25 ml/min. 3. Man 5 GN 1 4. Man 6 GN 1 5. Man 7 GN 1 6. Man 8 GN Man 9 GN _00 3-5O l,,,, i w i l i ' i i i i I i i, j i, l i i i l, l j i I,, i, i i, i i i, _ l i, 0 50 _00 _ Minutes
9 Hydrazine released ovomucoid DETECTION: UV at 200 nm oligosaccharides INJECTION:: 40 mg of mixture COLUMN: Prep Glyco-Pak TM N (22 x 600 mm) ELUENT: Acetonitrile/Water (70:30) FLOWRATE: 7.25 ml/min tn > '_ X I ' I ' I ' I ' t x 102 minut.es
10 Loading optimization study on a prototype Glyco-PakTMII DEAE in a glass AP column Purification of sialylated oligosaccharides from fetuin
11 In order to scale the sample load to larger columns for preparative isolation work, a loading study must be done on an ana!ytical column. Illustrated (Figures 5-8) is a loading optimization study utilizing fetuin oligosaccharides on a prototype Glyco-Pak Ii DEAE_in a glass AP column (10 x 100 mm). Figure 5 shows the analytical separation at 150 _g sample load. Figures 6 and 7 show sample loads of 5 and 10 mg respectively, with the 10 mg load showing signs of overload but still maintaining an acceptable separation. Figure 8 shows a sample overload of 50 mg with loss of resolution. From this study it is now known that a 10 mg load on the 10 x 100 mm column can be scaled to a 40 mg load on the 20 x 100 mm column and to 250 mg on the 50 x 100 mm column.
12 mv Hydrazine released fetuin oligosacch_llwes FLOW RATE: 1.6 ml/min COLUMN: Prototype Glyco-Pak TM II DEAE in a glass AP DETECTION: UV at 220 nm column (10 x 100 mm) INJECTION: 5 mg of mixture ELUENTA: Water PEAK ID'S: 1. Mono-sialylated ELUENTB: 100 mm KH2PO4, ph Di-sialylated GRADIENT: Hold for 5 minutes at 0 mm B, then linear from 3. Tri-sialylated 2 mm B to 50 mm B for 60 minutes, hold for 4. Tetra-sialylated 3O0 30 minutes Penta-sialylated 25O 2OO _ J - 0 ' ' ' ' ' ' ' ' ' I ' ' ' ' ' _ ' ' ' I ' ' I,,,,,, I ' ' ' ' ' ' " ' ' I ' ' ' ' ' ' Minutes
13 Hydrazine released fetuinoligosac( FLOW RATE: 1.6 ml/min C(_UMN: Prototype Glyco-Pak TM II DEAE in a glass AP DETECTION: UV at 210 nm column (10 x 100 mm) INJECTION: 150 lu.g of mixture ELUENTA: Water PEAK ID'S: 1. Mono-sialylated ELUENT B: 100 mm KH2PO4, ph Di-sialylated GRADIENT: Hold for 5 minutes at 0 mm B, then linear from 3. Tri-sialylated 2 mm B to 50 mm B for 60 minutes, hold for 4. Tetra-sialylated minutes m _- rv L_ l 0 ' ' ' ' ' ' ' ' ' I ' ' ' ' ' ' ' ' ' I ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' 1 ' "' ' ' ' 0 PO GO Minutes
14 SAMPLE: Hydrazine released fetuin oligosaccharides FLOW RATE: 1.6 ml/min COLUMN: Prototype Glyco-Pak TM II DEAE in a glass AP DETECTION: UV at 220 nm column (10 x 100 mm) INJECTION: 10 mg of mixture ELUENTA: Water PEAK ID'S: 1. Mono-sialylated ELUENTB: 100 mm KH2PO4, ph Di-sialylated GRADIENT: Hold for 5 minutes at 0 mm B, then linear from 3. Tri-sialylated 2 mm B to 50 mm B for 60 minutes, hold for 4. Tetra-sialylated 30 minutes. 5. Penta-sialylated mv 1000 B 800
15 SAMPLE: Hydrazine released fetuin oligosaccharides FLOW RATE: 1.6 ml/min COLUMN: Prototype Glyco-Pak TM II DEAE in a glass AP DETECTION: UV at 220 nm column (10 x 100 mm) INJECTION: 50 mg of mixture ELUENTA: Water PEAK ID'S: 1. Mono-sialylated ELUENT B: 100 mm KH2PO4, ph Di-sialylated GRADIENT: Hold for 5 minutes at 0 mm B, then linear from 3. Tri-sialylated 2 mm B to 50 mm B for 60 minutes, hold for 4. Tetra-sialylated 30 minutes. 5. Penta-sialylated 2200 k _ _ _ 2 4 _ BOO - I oo- /%/ 0 ',,,,,,,, I,,,',,,,,, I,,,,,, ' ' ' I '-_-_ ' ' ' ' ' ' I ''r-' _ "j-' Minutes
16 Illustrated is the isolation and purification of chitin oligosaccharides (Figures 9-15) utilizing a multiple chromatographic separation mode approach. UltrahydrogeFMDP ( aqueous size exclusion chromatography) and Glyco-PakTM N (acetonitrile/water partition chromatography) columns have been used to evaluate chitin samples prior to and after purification on a Prep Glyco-Pak N column. Figures 9 and 10 respectively show the separation of crude chitin and a fraction (Fraction A) of the higher oligomers using a bank of three Ultrahydrogel DP columns and on an analytical Glyco-Pak N column. With the Ultrah]drogel columns, the larger oligomer elutes first (Peak 6 is a larger oligomer than Peak 5). With the Glyco-Pak N column the reverse is true, the larger oligomer (Peak 6) elutes later than the smaller oligomer (Peak 5). Because the resolution of the oligomers is best on the Giyco-Pak N column, 8 mg of Fraction A chitin oligomers were purified on a Prep Glyco-Pak N column (Figure 11). Inorder to determine the purity of the materials isolated, Figures show the analysis of the fractions pooled to represent peaks 5 and 6 on the Ultrahydrogel DP columns and on the Glyco-Pak N column. Figure 12 shows that on the Ultrahydrogei DP columns, Peak 6 appears to be pure. However, the analysis of Peak
17 6 on the Glyco-Pak N column (Figure 13) shows that there is a small amount of Peak 5 still present. Figure 14 shows the analysis of Peak 5 with the Ultrahydrogel DP columns which is still contaminated with a small quantity of a larger oligomer (possibly Peak 6). Figure 15 shows that Peak 5 is also contaminated with a small quantity of a smaller oligomer (present as a shoulder). This multiple column approach, which utilizes two different selectivities (size exclusion and partition), illustrates the strength of using more than one chromatographic mode for the fractionation and purification of oligosaccharides prior to structure elucidation.
18 Preparative isolation of chitin oligosaccharides
19 Chitin Oligosaccharides 15 COLUMN: UItrahydrogelTM DP (3 columns-7.8 x 300 mm) ITIV ELUENT: Water _ FLOW RATE: 1 ml/min. :I0 COL. TEMP.: 50 C DETECTION: R I SENSITIVITY: 16 - INJECTION: 100 ug of mixture 0 I I 1 I I AU COLt..IE_: Glyco-PakTM N (7.8 x 300 ram) ELUENT: Acetonitrile/Water (72:28) 0 15 FLOW RATE: 1 ml/min. DETECTION: UV at 200 nm INJECTION: 150 ug of mixture 0 05 I I I-- 0 O0 ' I' ''I '''_... I... _''I '''l''l_' Minutes
20 Chitin Olig)saccharides Fraction A S 15 mv C(1Ub_: UltrahydrogelTM DP (3 columns-7.8 x 300 ram) - ELUENT: Water _.0 FLOWRATE: 1 ml/min. COL TEMP.: 50 C DETECTION: RI SENSITIVITY: INJECTION: 60 ug of mixture J 8...,) ^ 0 i -- i I I COLUIVlN: Glyco-PakTM N (7.8 x 300 mm) AU ELUENT: Acetonitrile/Water (72:28) - DEFECTION: UV at 200 nm 0.2 (_ FLOW INJECTION: RATE: mllmin. ug of mixture -- 4!,,, 0.0 ' ' -... I ' ' ' I ' I *, _,,,, I ','"t" 'b,,, ',,, I ' ' ' ' Hinutes
21 Chitin Oligosaccharides Fraction A DETECTION: UV at 200 nm COLUMN: Prep Glyco-Pak TMN (22 x 600 mm) INJECTION:: 8 mg of mixture ELUENT: Acetonitrile/Water (72:28) FLOWRATE: 7.25 ml/min..c I 1400 I mv J200 JO00 6 8OO 6OO _00 _ Minutes
22 C(_UMN: Ultrahydrogel m DP (3 columns-7.8 x 300 mm) DETECTION: RI ELUENT: Water SENSITIVITY: 1 6 FLOW RATE: 1 ml/min. COL. TEMP.: 50 C mv '15 - SAMPLE: ChitinOligosaccharides INJECTION: 100pgof mixture 5 _ 0 i J w I b i _'b I mv _5 - SAMPLE: Chitin Oligosaccharides - Fraction A 10 INJECTION: 60 _g of mixture / 5 _..,/_. 0 _ I i _ i mv '15 '10 SAMPLE: Chitin Oligosaccharide - Peak 6 - INJECTION: 20 pg
23 COLUMN: Glyco-Pak TM N (7.8 x 300 mm) DETECTION: UV at 200 nm ELUENT: Acetonitrile/Water (72:28) FLOW RATE: 1 ml/min. AU A SAMPLE: Chitin Oligosaccharides 0 _0-0:15 005,1 Jl _ J_ INJECTION: 150 lug of mixture i 0 00 i i, _ I AU / 0.3 SAMPLE: Chilin Oligosaccharides - Fraction A _ i i AU 0 _2-0 _.0 SAMPLE: Chitin Oligosaccharide - Peak INJECTION: 20 pg 0 O oo-k j, k 0 O0 I I ' I I-- I... "_#'--_l!... " '... ''"1 '''... I I I 1' _00
24 .i : Ultrahydrogel Water TM DP (3 columns-7.8 x _)mm) DETECTION: SENSITIVITY: RI 1 6 FLOW RATE: 1 ml/min. COL. TEMP.: 50 C mv 15 - SAMPLE: ChilinOligosaccharides INJECTION: 100lig of mixture A -, 0 i i i i 'l i i i mv _5 SAMPLE: Chitin Oligosaccharides - Fraction A '10 - INJECTION: 60 lig of mixture mvi A 15 SAMPLE: Chitin Oligosaccharide - Peak 5 _0 INJECTION: 20 p.g 0... I... I"''''_...!... ",i _,, '1-''... 'F'I,,-,'_... I,,,'v"... i _,,,'_ _0 _ Minutes
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