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Unit conversions in chromatography
UV cutoff of mobile phase aditives
| Aditivum | UV Cutoff (nm) |
| Acetic acid, 1 % | 230 |
| Ammonium acetate, 10 mM | 205 |
| Ammonium carbonate, 10 mM | 190 |
| Ammonium Hydrogenphosphate, 50 mM | 205 |
| CAPS 3-(cyklohexylamino)ethan sulfonic acid, 0,1 % | 215 |
| EDTA, 1 mM | 190 |
| Hydrochloric acid, 0,1 % | 190 |
| Kalium Hydrogenphosphate, 10 mM | 190 |
| Kalium Dihydrogenphosphate, 10 mM | 190 |
| MES 2-(N-morfolino)ethan sulfonic acid, pH 6,0, 10 mM | 215 |
| Natrium acetate, 10 mM | 205 |
| Natrium citrate, 10 mM | 225 |
| Natrium dodecylsulfate, 10 mM | 190 |
| Natrium formate, 10 mM | 200 |
| Natrium hexansulfonic acid, 5 mM | 225 |
| TEA, (triethylamine), 1% | 235 |
| TFA, (trifluoroacetic acid), 0,1 % | 190 |
| Tetrabutylammonium dihydrogen phosphate, 5 mM | 200 |
| TRIS HCl (Tris(hydroxylmethyl)aminomethan), pH 7,0, 20 mM | 202 |
| TRIS HCl (Tris(hydroxylmethyl)aminomethan), pH 8,0, 20 mM | 212 |
Unit conversions and tables
Unit conversions:
- Useful tables in chromatography
- Acid and bases pK used as an additivum for mobilne phases in HPLC
- UV cutoff of mobile phase components
- Column selection based on injection voluem and column capactiy
- Capillary choise for different flow rates
- ChromShell calculator (WE PREPARE)
- Pressure prediction in dependance of particle size, column length and diameter
- Viskosity of solvent mixtures
- Particle size conversion
Estimation of pressure on HPLC columns depending on particle size, diameter and column length
| Particle size (µm) | ID (mm) | Flow (ml/min) | Pressure 150mm* (PSI) | Pressure 250mm* (PSI) |
| 3 | 4.6 | 0.5 – 1.0 | 1000 | 1500 |
| 5 | 3.0 | 0.4 – 0.8 | 750 | 1250 |
| 5 | 4.6 | 1.0 - 2.0 | 700 | 1100 |
| 5 | 10.0 | 5.0 - 10.0 | 750 | 1250 |
| 10 | 4.6 | 2.0 - 5.0 | 400 | 600 |
| 10 | 10.0 | 10.0 - 20.0 | 500 | 800 |
| 10 | 21.2 | 20.0 - 40.0 | 300 | 500 |
* When the flow rate is at the lower limits of the recommended flow rates
Choice of capillaries for different flow rates
| id | Flow | Color | FROM |
| 0.13 mm | Up to 2.0 ml/min | Red | 1/16" |
| 0.18 mm | Up to 5.0 ml/min | Yellow | 1/16" |
| 0.25 mm | Up to 20 ml/min | Blue | 1/16" |
| 0.50 mm | Up to 50 ml/min | orange | 1/16" |
| 0.75 mm | Up to 100 ml/min | Green | 1/16" |
| 1.0 mm | Up to 200 ml/min | Grey | 1/16" |
| 1.59 mm | Up to 500 ml/min | 1/8"". | |
| 2.40 mm | Up to 1000 ml/min | 1/8"". |
Inches to mm - inch x 25.4 = mm
Feet per meter - feet x 0.3048 = meter
Column pressure at a flow rate of 1 ml/min
P = 2.1 xdx 10.13 xh / h 2 x vp 2
P – pressure (MPa)
L – column length in mm
h - dynamic viscosity (for water = 1)
d – internal diameter of the column in mm
vp – particle size in µm
The pressure on the 4.6 x 250 mm, 5 um column will be approx. 100 bar at a flow rate of 1.0 ml/min
Choice of column depending on the injection size and its capacity
| ID (mm) | Injection value (µl) | Column Capacity (mg) | Flow rate (ml/min) |
| 4,6 | 5 - 100 | 1 | 0,5 – 2,0 |
| 10 | 100 - 1000 | 5 | 4,0 – 15,0 |
| 21,2 | 1000 - 5000 | 20 | 10 – 50 |
| 30 | 2000 – 10 000 | 40 | 40 – 100 |
| 50 | 5000 – 20 000 | 120 | 100 – 300 |
| 100 | 10 000 – 50 000 | 500 | 400 - 1000 |
pK of acids and bases used as an additive for HPLC mobile phases
pKa of acidic buffers at HPLC for mobile phase preparation
| Acidic buffer | Temperature (°C) | pK1 | pK2 | pK3 | |
| ACES 2-[(2-amino-2-oxoethyl)amino]ethan sulfonic acid | 20 | 6.9 | - | - | |
| Acetic acid | 25 | 4.8 | - | - | |
| Boric acid | 20 | 9.1 | 12.7 | 13.8 | |
| CAPS 3-(cyklohexylamino)ethan sulfonic acid | 20 | 10.4 | - | - | |
| Citric acid | 25 | 3.1 | 4.8 | 6.4 | |
| Formic acid | 20 | 3.8 | - | - | |
| Glycine | 25 | 2.3 | 9.6 | - | |
| Glycylglycine | 20 | 8.4 | - | - | |
| HEPES N-2-hydroxyethylpiperazine-N'-2-ethan sulfonic acid | 20 | 7.6 | - | - | |
| Imidazole | 20 | 7.0 | - | - | |
| MES 2-(N-morfolino)ethan sulfonic acid | 20 | 6.2 | - | - | |
| MOPS 3-(N-morfolino)propan sulfonic acid | 20 | 7.2 | - | - | |
| Oxalic acid | 25 | 1.3 | 4.3 | - | |
| Phosphoric acid | 25 | 2.1 | 7.2 | 12.7 | |
| TES 2-[tris(hydroxymethyl)methyl]aminoethane sulfonic acid | 20 | 7.5 | - | - | |
| Trifluoroacetic Acid | 25 | 0.3 | - | - | |
| Tricine N-[tris(hydroxymethyl)methyl]glycine | 20 | 8.2 | - | - | |
| TRIS Tris(hydroxylmethyl) aminomethane | 20 | 8.3 | - | - | |
| pKb of Bases at HPLC for mobile phase preparation | |||||
| Bases | Temperature (°C) | pK1 | pK2 | pK3 | |
| Ammonia | 25 | 9.3 | - | - | |
| Diethylamine | 20 | 11.1 | - | - | |
| Dimethylamine | 25 | 10.7 | - | - | |
| Ethylamine | 20 | 10.8 | - | - | |
| Ethylendiamine | 20 | 10.1 | 7.0 | - | |
| Methylamine | 25 | 10.7 | - | - | |
| Morfoline | 25 | 8.3 | - | - | |
| Triethylamine (TEA) | 18 | 11.0 | - | - | |
| Trimethylamine | 25 | 9.8 | - | - | |
Note: The pH range for which the given buffer is suitable is in the range of pK ± 1. The UV Cutoff of the used buffer must also be taken into consideration (table 2).
Solvent viscosity
VIscosity dependance on solvent mixture composition
| % of water | Viscosity (MeOH/water) | Viscosity (AcCN/water) |
|---|---|---|
| 0 | 0.65 | 0.35 |
| 10 | 0.95 | 0.50 |
| 20 | 1.20 | 0.55 |
| 30 | 1.60 | 0.70 |
| 40 | 1.75 | 0.80 |
| 50 | 1.90 | 0.90 |
| 60 | 1.80 | 1.00 |
| 70 | 1.75 | 1.05 |
| 80 | 1.65 | 1.10 |
| 90 | 1.40 | 1.05 |
| 100 | 1.00 | 1.00 |
Chromatography tables
Capillary selection for various flowrates
Column selection in dependance on injection volume and its capacity
Mobile phase aditivum UV cutoff
pKa of acidic buffers at HPLC for mobile phase preparation
pKb of Bases at HPLC for mobile phase preparation
Pressure conversion
Pressure estimation in dependance on particle size, diameter, and column length
Solvent characteristics