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Chromatography
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).
Applications and support in UHPLC
Scale down (Method transfer from conventional HPLC to UHPLC)
Sample throughput (How do small particles increase the sample throughput?)
Scale down in UHPLC
Scale-down procedure from conventional HPLC to UHPLC requires optimizing columns selectivity and efficiency. As soon as we have finished this method development, we can perform a scale-down procedure. A few simple calculations can be used to determine equivalent run conditions. This article descibes them sequentially.
Adjusting Column Size
The first calculation determines the appropriate column length. Keeping the same column length while decreasing the particle size will increase the number of theoretical plates in that given column length. Therefore, column length can be shortened without losing resolution. By using Equation 1 and when adjusting the column length properly, we can maintain the same separation.
Adjusting Injection Volume
Once we have determined the proper column length, we can determine the appropriate injection volume. Decreasing the column internal diameter and length, decreases the overall column volume and sample capacity. Therefore, we must alter the injection volume as described in Equation 2. Please note that since overall column volume has decreased, it is important to match the sample solvent to the starting mobile phase composition. Mismatched sample solvents can cause irreproducible retention times, efficiencies, and even changes in selectivity.
Adjusting Flow Rate
Flow rate must be adjusted to maintain comparable linear velocity through a column with smaller internal diameter. Linear velocity is defined as the distance mobile phase travels over time, whereas flow rate is the volume of mobile phase that travels over time. To maintain the same linear velocity, which is important to maintain efficiencies, flow rates must be decreased as column internal diameter decreases. Also, since smaller particle sizes give rise to higher optimal linear velocities, isocratic flow rates should be calculated with particle size taken into account. Equation 3 can be used to simply and quickly estimate the adjusted flow rate needed for equivalent chromatography. It is also important to note that <2µm particle sizes are less affected by higher flow rates, and therefore faster flow rates can be used in isocratic systems without detrimental effects on peak efficiency.
Adjusting Time Program
Lastly, after we have determined the proper column length, injection volume, and flow rate, we can find the equivalent time needed for gradient or step elutions. As an analytical method is scaled down, the time program needs to also be scaled down to keep the phase interactions the same. Time can be adjusted using Equation 4.
Article referene: Rick Lake, Restek Coroporation
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
Carbotrap X
| Parameter | Characteristics |
|---|---|
| Sorbent strength/type | medium/strong carbon black |
| Specific surface area | approx. 240 m2/g |
| Approx. analyte volatility range | n-C3/4 to n-C6/7 (BP 50 to 150°C) |
| Example analytes | light hydrocarbons, 1,3-butadiene, benzene (for 2-week exposure) |
| Sorbent maximum temperature | > 400°C |
| Recommended conditioning temperature | 350 to 400°C |
| Recommended desorption temperature | 350 to 400°C ((but below conditioning temperature where possible) |
Further information
- hydrofobic
- minimal inherent artifacts (<0.1 ng)
- some activitity due to trace metals in the charcoal raw materials used for sorbent production
- friable - susceptible to fines formation when mechanically shocked. Avoid dropping tubes containing these sorbents
- compressable - overpacking can result in high impedance and adverseley affect pump sampling flows
- sorbent maximum temperature must not be exceeded as sorbent will breakdown and vapourise and may contaminate your flowpath
- 40/60 mesh is the recommended mesh size for sorbent tubes and traps
- recommended to re-pack tubes after 200 thermal cycles
Carbopack X
| Parameter | Characteristics |
|---|---|
| Sorbent strength/type | medium/strong carbon black |
| Specific surface area | approx. 240 m2/g |
| Approx. analyte volatility range | n-C3/4 to n-C6/7 (BP 50 to 150°C) |
| Example analytes | light hydrocarbons, 1,3-butadiene, benzene (for 2-week exposure) |
| Sorbent maximum temperature | > 400°C |
| Recommended conditioning temperature | 350 to 400°C |
| Recommended desorption temperature | 350 to 400°C ((but below conditioning temperature where possible) |
Further information
- hydrofobic
- minimal inherent artifacts (<0.1 ng)
- some activitity due to trace metals in the charcoal raw materials used for sorbent production
- friable - susceptible to fines formation when mechanically shocked. Avoid dropping tubes containing these sorbents
- compressable - overpacking can result in high impedance and adverseley affect pump sampling flows
- sorbent maximum temperature must not be exceeded as sorbent will breakdown and vapourise and may contaminate your flowpath
- 40/60 mesh is the recommended mesh size for sorbent tubes and traps
- recommended to re-pack tubes after 200 thermal cycles
Tenax GR
| Parameter | Characteristics |
|---|---|
| Sorbent strength/type | weak porous polymer |
| Specific surface area | approx. 35 m2/g |
| Approx. analyte volatility range | n-C7 to n-C30 (BP 100 to 450°C) |
| Example analytes | aromatic compounds (except benzene), apolar components with BP >100°C, polar components with BP >150°C, PAHs, PCBs |
| Sorbent maximum temperature | > 350°C |
| Recommended conditioning temperature | up to 325°C |
| Recommended desorption temperature | up to 300°C |
Further information
- hydrofobic
- low inherent artifacts (<1 ng)
- inert - suitable for labile compounds
- Tenax GR (graphitised form) is the best for PAHs and PCBs
- efficient desorption
- sorbent maximum temperature must not be exceeded as sorbent will breakdown and vapourise and may contaminate your flowpath
- 35/60 mesh is the recommended mesh size for sorbent tubes and traps
- recommended to re-pack tubes after 100 thermal cycles