Phone +420 274 021 211
About us
Contact
EN
Search
0 Compare
Add products to the comparison using the scales icon and here you can then compare their parameters.
User
0 Basket
Your basket is empty...
MENU
  1. Chromservis
  2. Chromatography
Category
Flash and Preparative HPLCGC/MS-TOF
    GC/MS/MS
      Glossary
        Hints and tipsMetAmino kitStationary phasesSyringesUHPLCUnit conversions

        Chromatography

        Solvent characteristics

        Solvent Polarity index Refractive index (20°C)

        UV (nm) cutoff

        (1 AU)

        		 Boiling point (°C)

        Viscosity

        (mPa.s; 20°C)

        		 Miscibility with water
        Acetic acid 6.2 1,372 230 118 1,26 100
        Acetone 5.1 1,359 330 56 0,32 100
        Acetonitrile 5.8 1,344 190 82 0,37 100
        Benzene 2.7 1,501 280 80 0,65 0,18
        Butylacetate 4.0 1,094 254 125 0,73 0,43
        n-Butanol 3.9 1,399 215 118 2,98 7,81
        Carbon Tetrachloride 1.6 1,466 263 77 0,97 0,08
        Chloroform 4.1 1,466 245 61 0,57 0,815
        Cyclohexane 0.2 1,426 200 81 1,00 0,01
        Ethylenechloride 3.5 1,444 225 84 0,79 0,81
        Methylenechloride 3.1 1,424 235 41 0,44 1,6
        Dimethylformamide 6.4 1,431 268 155 0,92 100
        Dimethylsulfoxide 7.2 1,478 268 189 2,00 100
        Dioxane 4.8 1,422 215 101 1,54 100
        Ethylacetate 4.4 1,372 260 77 0,45 8,7
        Ethanol 5.2 1,360 210 78 1,20 100
        Diethylether 2.8 1,353 220 35 0,32 6,89
        Heptane 0.0 1,387 200 98 0,39 0,0003
        Hexane 0.0 1,375 200 69 0,33 0,001
        Methanol 5.1 1,329 205 65 0,60 100
        Methylbutylether 2.5 1,369 210 55 0,27 4,8
        Methylethylketone 4.7 1,379 329 80 0,45 24
        Pentane 0.0 1,358 200 36 0,23 0,004
        n-Propanol 4.0 1,384 210 97 2,27 100
        Isopropanol 3.9 1,377 210 82 2,30 100
        Diisopropylether 2.2 1,368 220 68 0,37
        Tetrahydrofuran 4.0 1,407 215 65 0,55 100
        Toluene 2.4 1,496 285 111 0,59 0,051
        Trichlorethylene 1.0 1,477 273 87 0,57 0,11
        Water 9.0 1,333 200 100 1,00 100
        Xylene 2.5 1,500 290 139 0,61 0,018

        Dependence of the viscosity of the solvent mixture on its percentage composition

        % of water Viscosity (MeOH/water) Viscosity (ACN/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

        Chart

        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).

        Applications and support in UHPLC

        ChronoScale 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.

        CalculationAdjusting 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.

        CalculationAdjusting 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

        Viscosity of solvent mixtures

        Solvent characteristics