BÜCHI Labortechnik AG Loadability page 1
Load ability Loading Capacity Resolution Sample Solubility (injection) Conclusion 2 page 2
Loading Capacity(active sites/sample affinity) Competitor B Competitor C Vancomycin Frontal Loading Absorbance at 276 nm 4321 Competitor A Competitor D Breakthrough Point 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 45 Time (min.) Column Competitor D, C18 10µm Competitor C, C18 15µm Competitor B, C18 10µm Competitor A, C18 15-20µm 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 = 0.173 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
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
Loading Capacity(active sites saturation) Vydac 150HC C18 20µm 25 µg 50 µg 100 µg 250 µg 1000 µg 5 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 Time (min.) Sample Load (µg) Vydac 150HC C18 20µm Efficiency (plates) 25 2132 50 2149 100 1578 250 1118 1000 365 page 5
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
Loading capacity (Particle Sizes) C4 24-95% 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
Resolution Relation between resolution and loadability WhenΔR f increases the sample loadability increases 8 page 8
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
Injection(Analytical) 10 page 10
Injection(Overload) 11 page 11
Injection(Overload types) 12 page 12
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
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 0,2 0 2 4 6 volume Injection (ml) Peak 2-3 Peak 3-4 14 page 14
Loading capacity by injection volume (solvent strength) 15 page 15
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 3-4 16 page 16
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
Sandwich Injection (precipitation) 18 page 18
Solid loading 19 page 19
Solid loading (example) 500mg mix of a mono saccharide and glycosylated peptides. Coated on 3 gram celite 20 page 20
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