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1 BÜCHI Labortechnik AG Loadability page 1

2 Load ability Loading Capacity Resolution Sample Solubility (injection) Conclusion 2 page 2

3 Loading Capacity(active sites/sample affinity) Competitor B Competitor C Vancomycin Frontal Loading Absorbance at 276 nm 4321 Competitor A Competitor D Breakthrough Point Time (min.) Column Competitor D, C18 10µm Competitor C, C18 15µm Competitor B, C18 10µm Competitor A, C µm Loading Capacity 31 mg/ml 11 mg/ml 7 mg/ml 13 mg/ml The capacity of each material is calculated from the following equation: C Capacity = ([T (at 50%) t 0(uracil) ] x C vancomycin x F) V Column T (at 50%) = Frontal breakthrough time measured at 50% peak height minus 1 minute. C vancomycin = 1 mg/ml V Column = ml F (flow rate) = 0.2 ml/min t 0uracil determined from an injection of uracil with mobile phase A at 0.2mL/min. 3 page 3

4 Loading capacity (Surface Area) Effect on chromatography High surface area generally provides greater retention, capacity and resolution for separating complex, multi-component samples. Low surface area packings generally equilibrate quickly, especially important in gradient analyses. 4 page 4

5 Loading Capacity(active sites saturation) Vydac 150HC C18 20µm 25 µg 50 µg 100 µg 250 µg 1000 µg Time (min.) Sample Load (µg) Vydac 150HC C18 20µm Efficiency (plates) page 5

6 Loading capacity (Scaling Up) C4, 300Å, 4.6 x 250 mm, 5 µm 24-95% ACN in 0.1% TFA over 30 min. 2-fold increase in column inner diameter 4 to 5-fold increase in capacity 6 page 6

7 Loading capacity (Particle Sizes) C % ACN in 0.1% TFA over 30 min. at 1.5 ml/min. Peak widths much narrower with small particle materials at low sample loads. However, little difference in peak widths at high loads. Allows usage of larger particle sizes resulting in lower cost. 7 page 7

8 Resolution Relation between resolution and loadability WhenΔR f increases the sample loadability increases 8 page 8

9 Resolution (choices) Column packing material ph of eluens Temperature Selectivity triangle H- Donor VIII H- Acceptor II Ethanol IV Acetic acid I Diethylether III Tetrahydrofuran VI V ChloroformEthylacetate Dichloromethane Toluene VII Dipole 9 page 9

10 Injection(Analytical) 10 page 10

11 Injection(Overload) 11 page 11

12 Injection(Overload types) 12 page 12

13 Injection(test) In this part of the comparison 2 types of tests are performed : Loading capacity by volume of injection Loading capacity by mass of sample A standard Reveleris system was used with the following conditions : Solvent : Gradient heptane / ethyl acetate 0 to 25 % in 15 CV. Flow : 36ml/min for all other cartridges Detection : ELSD + UV1 (254nm) + UV2 (280nm) Sample: Phtalate test mix in Heptane toluene (5g) dimethylphtalate (10g) diethylphtalate (15g) dibutylphtalate (20g) 13 page 13

14 Loading capacity by injection volume Mass of sample Injection Volume 1.4g 2ml 1.4g 4ml 1.4g 8ml Resolution as function from injection volume Resolution 1,6 1,4 1,2 1 0,8 0,6 0,4 0, volume Injection (ml) Peak 2-3 Peak page 14

15 Loading capacity by injection volume (solvent strength) 15 page 15

16 Loading capacity by sample mass 2 1,8 1,6 1,4 Resolution as function from sample mass Resolution 1,2 1 0,8 0,6 0,4 0,2 0 0,7 1,4 2,1 2,8 Sample load (gram) Peak 2-3 Peak page 16

17 Solutions to overcome injection limitation o Direct injection on column. o The injection valves are bypassed. o Sandwich injection method o Solid loading 17 page 17

18 Sandwich Injection (precipitation) 18 page 18

19 Solid loading 19 page 19

20 Solid loading (example) 500mg mix of a mono saccharide and glycosylated peptides. Coated on 3 gram celite 20 page 20

21 Conclusion Load abilities limitation are physical Solubility Affinity Selectivity Resolution Precipitation No controlable : It is impossible to overcome Physical limitation controlable: There are a lot of parameters to play with -Solvents (ph, selectivity triangle) -packing material (selectivity, service area, particle, active sites etc.) -injection method 21 page 21

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