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Procedures: The Module

  1. Use the run parameters outlined below to obtain triplicate runs of a standard containing both a neutral marker (for example, dimethylformamide or mesityl oxide) and atenolol. Report the migration times, peak height, and calculated theoretical plates for the neutral marker and atenolol for each run. Calculate the mean, standard deviation (SD) of the mean, and relative standard deviation (RSD) of the mean for each parameter. You may use the table below to report your data.

    Preparation of capillary: Assuming this is the first run of the day, the capillary should be flushed as follows: 30-min, 20 psi flush with 0.1 N NaOH, followed by 15-min, 20 psi flush with deionized water, followed by a 30-min, 20-psi flush with running buffer. This flushing protocol is necessary only at the beginning of the day. If exercises 1-6 are completed within a single lab period, repeating this initial capillary flush should not be necessary.

    Sample: 0.65 millimolar (0.005 % v/v) dimethylformamide, 75 micromolar atenolol in 25 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES) buffered at pH 7.0.

    Injection parameters: 1 second injection at 2 psi pressure

    Run conditions: running buffer 25 mM TES pH 7.0, 25 micron i.d. fused silica capillary, 42.8 cm total length, 32.9 cm to detection window, separation voltage: 15 kV, range 0.001, rise time 0.3 seconds, 220 nm

    Injection parameters: 1 second injection at 2 psi pressure
    Atenolol Trial 1 Trial 2 Trial 3 Mean

    Peak Height

    0.3446 0.3738 0.3495 0.356

    Migration Time (sec)

    77.19 77.55 77.43 77.39

    σ (sec)

    0.620 0.610 0.550  
    Plates (x 103) 15.500 16.162 19.820 17.2
    Neutral Marker        

    Peak Height

    0.5154 0.50626 0.49369 0.505

    Migration Time (sec)

    109.3 109.48 109.27 109.35

    σ (sec)

    1.010 1.010 0.9232  
    Plates (x 103) 11.711 11.750 14.009 12.5

     

  2. Use the run parameters outlined below to obtain triplicate runs of a standard containing both a neutral marker and atenolol. Report the migration times, peak height, and calculated theoretical plates for the neutral marker and atenolol for each run. Calculate the mean, SD and RSD for each parameter. You may use the table below to report your data.

    Sample: 0.65 millimolar (0.005 % v/v) dimethylformamide, 75 micromolar atenolol in 25 mM TES pH 7.0.

    Injection parameters: 1 second injection at 4 psi pressure

    Run conditions: running buffer 25 mM TES pH 7.0, 25 micron i.d. fused silica capillary, 42.8 cm total length, 32.9 cm to detection window, separation voltage: 15 kV, range 0.001, rise time 0.3 seconds, 220 nm.

    Injection parameters: 1 second injection at 4 psi pressure
    Atenolol Trial 1 Trial 2 Trial 3 Mean

    Peak Height

    0.33242 0.4253 0.40625 0.388

    Migration Time (sec)

    75.789 76.7 77.285 76.59

    σ (sec)

    1.1813 1.2279 1.1416  
    Plates (x 103) 4.116 3.902 4.583 4.2
    Neutral Marker        

    Peak Height

    0.59611 0.60641 0.58779 0.5968

    Migration Time (sec)

    107.07 107.48 108.75 107.77

    σ (sec)

    1.6278 1.8318 1.7917  
    Plates (x 103) 4.326 3.443 3.684 3.82

     

  3. Use the run parameters outlined below to obtain triplicate runs of a standard containing both a neutral marker and atenolol. Report the migration times, peak height, and calculated theoretical plates for the neutral marker and atenolol for each run. Calculate the mean, SD and RSD for each parameter. You may use the table below to report your data.

    Sample: 0.65 millimolar dimethylformamide, 75 micromolar atenolol in 25 mM TES pH 7.0.

    Injection parameters: 1 second injection at 1.5 psi pressure

    Run conditions: running buffer 25 mM TES pH 7.0, 25 micron i.d. fused silica capillary, 42.8 cm total length, 32.9 cm to detection window, separation voltage: 15 kV, range 0.001, rise time 0.3 seconds, 220 nm

    Injection parameters: 1 second injection at 1.5 psi pressure
    Atenolol Trial 1 Trial 2 Trial 3 Mean

    Peak Height

    0.34328 0.30514 0.31077 0.320

    Migration Time (sec)

    78.266 77.564 78.339 78.06

    σ (sec)

    0.50504 0.45251 0.49427  
    Plates (x 103) 24.016 29.381 25.120 26.2
    Neutral Marker        

    Peak Height

    0.45072 0.40995 0.45533 0.439

    Migration Time (sec)

    110.36 109.69 110.79 110.28

    σ (sec)

    0.82552 0.76076 0.86963  
    Plates (x 103) 17.872 20.789 16.231 18.3

     

  4. Use the run parameters outlined below to obtain triplicate runs of a standard containing both a neutral marker and atenolol. Report the migration times, peak height, and calculated theoretical plates for the neutral marker and atenolol for each run. Calculate the mean, SD and RSD for each parameter. You may use the table below to report your data.

    Sample: 0.65 millimolar dimethylformamide, 75 micromolar atenolol in 25 mM TES pH 7.0.

    Injection parameters: 2 second injection at 2 psi pressure

    Run conditions: running buffer 25 mM TES pH 7.0, 25 micron i.d. fused silica capillary, 42.8 cm total length, 32.9 cm to detection window, separation voltage: 15 kV, range 0.001, rise time 0.3 seconds, 220 nm

    Injection parameters: 2 second injection at 2 psi pressure
    Atenolol Trial 1 Trial 2 Trial 3 Mean

    Peak Height

    0.39943 0.40209 0.40322 0.4016

    Migration Time (sec)

    77.054 79.18 78.092 78.1

    σ (sec)

    1.1705 1.1353 1.132  
    Plates (x 103) 4.334 4.864 4.759 4.7
    Neutral Marker        

    Peak Height

    0.62067 0.6374 0.62069 0.6263

    Migration Time (sec)

    109.87 111.73 110.4 110.67

    σ (sec)

    1.7754 1.7065 1.8522  
    Plates (x 103) 3.830 4.287 3.553 3.89

     

  5. Use the run parameters outlined below to obtain triplicate runs of a standard containing both a neutral marker and atenolol. Report the migration times, peak height, and calculated theoretical plates for the neutral marker and atenolol for each run. Calculate the mean, SD and RSD for each parameter. You may use the table below to report your data.

    Sample: 0.65 millimolar dimethylformamide, 75 micromolar atenolol in 25 mM TES pH 7.0.

    Injection parameters: 4 second injection at 2 psi pressure

    Run conditions: running buffer 25 mM TES pH 7.0, 25 micron i.d. fused silica capillary, 42.8 cm total length, 32.9 cm to detection window, separation voltage: 15 kV, range 0.002, rise time 0.3 seconds, 220 nm

    Injection parameters: 4 second injection at 2 psi pressure
    Atenolol Trial 1 Trial 2 Trial 3 Mean

    Peak Height

    0.21345 0.22416 0.2202 0.2193

    Migration Time (sec)

    76.682 76.162 75.8220 76.22

    σ (sec)

    2.0975 2.2539 2.2092  
    Plates (x 103) 1.337 1.142 1.178 1.2
    Neutral Marker        

    Peak Height

    0.32948 0.34387 0.34916 0.341

    Migration Time (sec)

    108.39 107.84 107.24 107.82

    σ (sec)

    3.3362 3.5499 3.6671  
    Plates (x 103) 1.056 0.923 0.855 0.94

     

  6. Use the run parameters outlined below to obtain a single run of a standard containing both a neutral marker and atenolol. Report the migration times, peak height, and calculated theoretical plates for the neutral marker and atenolol for each run.

    Sample: 0.65 millimolar dimethylformamide, 75 micromolar atenolol in 25 mM TES pH 7.0.

    Injection parameters: 2 second injection at 4 kV applied voltage (normal polarity, no pressure!)

    Run conditions: running buffer 25 mM TES pH 7.0, separation voltage: 15 kV, range 0.001, rise time 0.3 seconds, 220 nm

    2 s, 4 kV
    Atenolol Trial 1

    Peak Height

    0.21544

    Migration Time (sec)

    77.304

    σ (sec)

    0.41192
    Plates (x 103) 35.219
    Neutral Marker  

    Peak Height

    0.23153

    Migration Time (sec)

    109.15

    σ (sec)

    0.64349
    Plates (x 103) 28.772

     

  7. Use your findings from the exercises outlined in steps 1-5 to complete the table below. What conclusions can you draw from these studies? What injection protocol is best suited for analyses of 75 micromolar atenolol?
    Injection Conditions 4s, 2psi 2s, 2psi 1s, 4psi 1s, 2psi 1s, 1.5psi

    Atenolol

             

    Height, Mean

    0.219 0.402 0.39 0.36 0.32

    Time (s), Mean

    76.2 78 76.6 77.4 78.1
    Plates 1000 5000 4000 17000 26000

    Neutral Marker

             

    Height, Mean

    0.34 0.626 0.597 0.50 0.43

    Time (sec), Mean

    107.8 110.7 107.8 109.4 110.3
    Plates 900 3900 3800 12000 18000

    The plate count is highest when the total injection volume is low.

  8. Independent Excursion: As you can see from this exercise, the injection protocol can have a marked effect on various aspects of the separation. Many other injection protocol, concerns and criteria are important to other applications of capillary electrophoresis. For instance, in addition to hydrodynamic injection (forcing sample on using pressure), injections may also be facilitated by voltage and siphoning. The result produced in step 6 of this lab exercise, might lead you to devise another set of experiment designed to find optimal injection parameters using electrophoretic injection. Difference in the ionic strength of the sample and running buffer can also effect the separation. Below you will find two proposed experiments that might provide insight in these two areas. After you have had time to ponder the experiment performed in steps 1-6, you may attempt the independent experiments below, or design your own experiment.

    Injection facilitated by siphoning:

    Elevate the anodic buffer reservoir and the separation capillary such that the liquid line (top of vial) and capillary end in are 8 cm higher in the anodic reservoir relative to the cathodic reservoir. Determine the mean value for migration time (n=2) for both a neutral marker and to lmetin. Repeat this exercise with the liquid line (top of vial) and capillary end are at identical heights in both the anodic and cathodic reservoirs.

    Determination of analyte in matrix of ionic strength different from running buffer*

    Use identical pressure-based injection and run conditions for the samples outlined below. Determine the mean values for migration time, height and theoretical plate count for triplicate runs of each of the solutions listed below. Be sure to report the uncertainty in your answers.

    (A) neutral marker and 100 micromolar mesityl oxide, and 300 micromolar tolmetin dissolved in deionized water

    (B) neutral marker and 300 micromolar tolmetin dissolved in background electrolyte (25 mM TES pH 7.0)

    (C) neutral marker and 300 micromolar tolmetin dissolved in background electrolyte (25 mM TES pH 7.0) that also contains 500 mM sodium chloride

    *Note: this exercise may be attempted using voltage-based injections (electrophoretic) or siphoning.

  9. Outline the experimental strategy to determine the concentration of atenolol in an aqueous solution.

    The concentration of atenolol in an aqueous solution can be determined by using a calibration curve. A calibration curve can be generated using a set of atenolol standards that relates the absorbance versus peak height or absorbance versus peak area. The calibration curve will only be valid for the experimental conditions under which the measurements were made (range, wavelength, rise time, injection time, injection pressure, separation voltage). If one of the parameters is changed, such as the wavelength, then a new calibration curve would have to be generated before measuring the concentration of atenolol in an unknown sample. If the curve is fit using linear regression, then the concentration range must be within the linear range of the method. You may have to determine the linear range of the method before making your first calibration curve. Finally, due to drifting of the lamp source and because the fused silica capillary must be flushed periodically, the measurements of the standards and the unknown sample should occur as timely as possible to prevent the introduction of time-dependent error. Below is a 3-point calibration curve for atenolol. In this case the sample contained 125 micromolar atenolol. Based on the response for the unknown (0 .66536 mAu) and error in the calibration curve, the concentration is 130 ± 7 micromolar.

  10. Lab Practical
    Through a faculty mentor or collaborator, you should arrange to receive an unknown solution containing either naproxen or atenolol diluted in background electrolyte at a concentration above the limit of quantification for your capillary electrophoresis system. Once you receive the unknown solution, you are to determine the analyte composition and concentration in the solution. Your final report should include pertinent data and a clear explanation of your results. When you report your final value, be sure to include uncertainty. You should have access to standard solutions as necessary.