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  1. Chromservis
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Chromatography

Flash and Preparative HPLCGC/MS-TOF
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        Unit conversions

        Unit Conversions

        HPLC capillaries volume chart

        ID (mm) ID (inch) µl/cm µl/inch ID (mm) ID (inch) µl/cm µl/inch
        0.050 0.002" 0.02 0.05 1.00 0.040" 7.85 20.59
        0.064 0.0025" 0.03 0.08 1.40 0.055" 15.39 38.93
        0.075 0.003" 0.04 0.12 1.52 0.060" 18.15 46.33
        0.10 0.004" 0.08 0.21 1.59 0.062" 19.86 49.47
        0.13 0.005" 0.13 0.32 1.65 0.065" 21.38 54.38
        0.17 0.0067" 0.23 0.58 1.70 0.067" 22.70 57.78
        0.18 0.007" 0.25 0.63 1.78 0.070" 24.88 63.06
        0.25 0.010" 0.49 1.29 2.00 0.079" 31.42 80.32
        0.38 0.015" 1.13 2.90 2.10 0.083" 34.64 88.66
        0.50 0.020" 1.96 5.15 2.16 0.085" 36.64 92.99
        0.75 0.030" 4.42 11.58 2.40 0.094" 45.24 113.72

        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 ConversionUnit conversions:

        Particle size conversion

        Mesh size (No.) Metric size (µm) Nominal opening (inches)
        40 420 0.0165
        45 354 0.0139
        50 297 0.0117
        60 250 0.0098
        70 210 0.0083
        80 177 0.0070
        100 149 0.0059
        120 125 0.0049
        140 105 0.0041
        170 88 0.0035
        200 74 0.0029
        230 63 0.0025
        270 53 0.0021
        325 44 0.0017
        400 37 0.0015

        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).