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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).