ZORBAX Reversed-Phase HPLC Column Selection Flow Chart
For small and large molecules
Most chromatographers use reversed-phase HPLC as one of their key analysis techniques. Reversed-phase HPLC can be used to analyze ionic and nonionic analytes. Therefore, this ZORBAX Column Selection flow chart will focus on reversed-phase columns. To more easily select a reversed-phase column for method development of small and large molecules, follow the outline below.
This flow chart provides information on choosing an initial column for method development of small molecule and protein and peptide samples, and includes decisions on bonded phase and column configuration. You can also download the chart as a PDF or download the entire Agilent ZORBAX Column Selection Guide.
Small Molecules
MW < 2000 |
Large Molecules
MW > 2000 |
| 80-120Å |
Packing Pore Size |
300Å |
First choice of a packing pore size is based on the size of molecules to be analyzed. Typical small molecules can diffuse easily in and out of standard 80-120Å pore packings, but peptides and proteins may not. For this reason, it is recommended to use 300Å pore-size packings (300SB) for isocratic or gradient separations of peptides and proteins. |
| Eclipse Plus C18 |
Starting Column Bonded Phase |
StableBond 300SB-C18 |
A C18 is recommended as the starting column bonded phase for most samples since it maximizes retention for moderately polar to non-polar compounds. Shorter chain phases should be considered if resolution cannot be optimized with a C18 phase or if you are analyzing larger proteins. |
| 4.6 mm |
Column Diameter |
4.6 mm |
Column diameter defines how much material (mass) you can inject on a column - the smaller the diameter, the less material that can be injected while maintaining good peak shape. Smaller diameter columns provide increased sensitivity and therefore are commonly used for LC/MS and sample limited applications. |
| Column Type |
Inner Diameter (mm) |
Sample Load (approx) |
Typical Flow Rate |
Increased Sensitivity |
Applications |
| Analytical |
4.6 |
0.1-1.5 mg |
0.5-3 mL/min |
|
Standard separations |
| Solvent Saver |
3.0 |
150-500 ug |
0.3-1.5 mL/min |
+ |
Save solvent, uses standard HPLC instrument |
| NarrowBore |
2.1 |
50-120 ug |
0.1-0.5 mL/min |
++ |
High sensitivity, limited sample, LC/MS, save solvent |
| Microbore |
1.0 |
10-50 ug |
10-100 µL/min |
++++ |
High sensitivity, limited sample, LC/MS |
| Capillary |
0.5, 0.3 |
1-10 ug |
1-15 µL/min |
+++++ |
Very high sensitivity, LC/MS, peptides and proteins |
| Nano |
0.1, 0.075 |
100-200 ng |
200-500 nL/min |
++++++ |
Very high sensitivity, LC/MS, peptides and proteins |
| Semi-Prep |
9.4 |
1-10 mg |
5-10 mL/min |
|
mg preparative separations |
| Preparative |
21.2 |
20-250 mg |
20-60 mL/min |
|
Hundreds of milligrams up to 1 gram |
Small Molecules
MW < 2000 |
Large Molecules
MW > 2000 |
| 5 µm |
Standard Porous Particle Size |
5 µm |
For conventional analyses of small and large molecules, the standard particle size is 5 µm. However, smaller particle sizes are available and provide higher efficiency and higher resolution. They are also available in short column lengths. |
|
Fast Analysis Particle Sizes |
| |
|
Rapid Resolution 3.5 µm
Rapid Resolution HT 1.8 µm
Rapid Resolution, 3.5µm particles provide 60% greater efficiency than 5µm particles while Rapid Resolution HT columns, 1.8µm particles provide 200% greater efficiency. Shorter columns can be used to significantly reduce analysis time of small molecules. |
Rapid Resolution 3.5 µm
Poroshell 5 µm
For peptides, Rapid Resolution, 3.5µm particles can be used with shorter columns for faster, high-resolution gradient analyses. But for proteins, Poroshell columns, with a solid core and porous shell, provide the fastest analysis. |
| 150 mm |
Column Length |
150 mm | |
|
A conventional column length is 150mm or 250mm for high resolution with 5µm particles. This is a good place to start a typical analysis because it will provide high resolution in a 20-30 minute analysis. For fast analysis, shorter columns (75mm and 50mm) with smaller particle sizes (3.5µm) should be used. |
For proteins and peptide digests, 150mm is also a good starting column length. The typical gradient time on a column of this length will be 30-60 minutes for a high-resolution analysis. For fast analysis, shorter columns (50mm) with smaller particles (3.5µm) should be used. |
| Starting Column Choices |
| Small Molecules |
Large Molecules |
|
Standard Analysis
Eclipse Plus C18
4.6 x 150 mm, 5 µm
PN 959993-902
|
Fast Analysis
Rapid Resolution
Eclipse Plus C18
4.6 x 75 mm, 3.5 µm
PN 959933-902
Rapid Resolution HT
Eclipse Plus C18
4.6 x 50 mm, 1.8 µm
PN 959941-902
|
Standard Analysis
300SB-C18
4.6 x 150 mm, 5 µm
PN 883995-902 |
Fast Analysis
Rapid Resolution
300SB-C18
4.6 x 50 mm, 3.5 µm
PN 865973-902
Poroshell
300SB-C18
2.1 x 75 mm, 5 µm
PN 660750-902
|
For additional method development guidelines, review the section on method development from low to high pH. |
|