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Chemistry LibreTexts

Procedures: The Module

  1. Outline the experiments you must do to determine the retention factor (capacity factor) for 4 NSAIDs. To determine the retention factor (k’) for four NSAIDs, several steps must be followed. First, the free-zone migration times for both the NSAIDs and a neutral marker (neutral, hydrophilic compound such as dimethylformamide) must be determined in triplicate. Next, the MEKC migration times for the NSAIDs, a neutral marker, and a micelle marker (neutral, highly hydrophobic compound such as n-decanophenone) must be determined in triplicate. All migration times should be determined using the same bare-fused silica capillary, and the length to the detection window (Lw), the total length (Lt), and separation voltage (Vsep) must be known. The run buffer should be the same concentration with respect to the buffering species (CAPs) and pH for both the free-zone and MEKC trials. From the free-zone runs, you should be able to determine the free-zone electrophoretic mobility for each NSAID (μeph_NSAID) using the following equation:

    μapparent_NSAID (CE) = [Lw x Lt] / (tNSAID(CE) x Vsep)

    μeof free zone = [Lw x Lt] / ( teof marker_DMF x Vsep)

    μeph_NSAID = μapparent_NSAID(CE) - μeof free zone

    From the MEKC runs, the electroosmotic mobility for each trial (μeof_MEKC) is determined using the following equation:

    μeof MEKC = [Lw x Lt] / (teof marker(MEKC) x Vsep)

    By combining these two terms, along with the migration times of the eof marker (teof (MEKC)), the NSAID (tNSAID (MEKC)), and the micelle marker (tn-dec_ (MEKC)), all of which are obtained from the MEKC trials, the retention factor (k’) is calculated for each NSAID using the equation below.

    \[k' = \dfrac{t_{tolmetin\_MEKC}\left(1+\dfrac{\mu_{eph\_tolmetin}}{\mu_{eof\_MEKC}}\right) - t_{eof\_MEKC}}{t_{eof\_MEKC}\left(1 - \dfrac{t_{tolmetin\_MEKC}}{t_{n−dec\_MEKC}}\right)}\]

    ttolmetin_MEKC = MEKC migration time tolmetin

    μeph_tolmetin = calculated CE electrophoretic mobility tolmetin

    μeof_MEKC = MEKC electroosmotic mobility (DMF)

    teof_MEKC = MEKC migration time DMF

    tn-dec_MEKC = MEKC migration time n-decanophenone

    The retention factor for each NSAID per trial should be determined in triplicate. The mean value for k’ for each NSAID should be reported. It is important to note that under optimized conditions, all four NSAIDs can be separated during a single MEKC trial, giving individual migration times for each NSAID. However, for the free-zone trials, each NSAID must be analyzed separately because of the similarity in migration times under free-zone conditions.

  2. Determine the retention factor (capacity factor) for flurbiprofen, naproxen, sulindac, and tolmetin. These values should be reported as mean values determined from triplicate runs (n = 3). For these runs, the background BGE is 25 mM CAPs, 100 mM SDS buffered to pH 10. You must use a ~25 micron inner diameter fused silica capillary ~42 cm in total length, ~32 cm to the window, 20,000 V.

    Free Zone Data
    Lw = 32.9 cm, Lt = 42.8 cm, Vsep = 20,000 V, pH 10 CAPS

      Migration Time μ eof (CE)

     

    Marker

    Trial

    1

     

    2

     

    3

    Trial

    1

     

    2

     

    3

    DMF 105.1 104.7 104.7 0.04019 0.04034 0.04034
      Migration Time μ app

     

    Analyte

    Trial

    1

     

    2

     

    3

    Trial

    1

     

    2

     

    3

    Tolmetin

    147.4 147.0 147.2 0.2867 0.2874 0.2870

    Naproxen

    152.8 153.1 152.8 0.2765 0.2759 0.2765

    Flurbiprofen

    152.3 151.8 152.0 0.2773 0.2784 0.2780
    Sulindac 141.5 139.5 140.5 0.2986 0.3028 0.3006
      μ eph          

     

    Analyte

    Trial

    1

     

    2

     

    3

     

    AVE

       

    Tolmetin

    -0.01153 -0.01160 -0.01164 -0.01159    

    Naproxen

    -0.01254 -0.01275 -0.01269 -0.01266    

    Flurbiprofen

    -0.01246 -0.01250 -0.01254 -0.01250    
    Sulindac -0.01033 -0.01006 -0.01028 -0.01022    

     

    MEKC DATA
    Lw = 32.9 cm, Lt = 42.8 cm, Vsep = 20,000 V, pH 10 CAPS 100 mM SDS
      Migration Time μ eof (MEKC)  

     

    Marker

    Trial

    1

     

    2

     

    3

    Trial

    1

     

    2

     

    3

     

    DMF

    126.0 125.9 126.0 0.03354 0.03356 0.03353  
    n-dec 353.4 354.8 355.9        
      Migration Time k'  

     

    Analyte

    Trial

    1

     

    2

     

    3

    Trial

    1

     

    2

     

    3

     

    AVE

    Tolmetin

    196.9 196.6 197.4 0.0523 0.0511 0.0569 0.0534

    Naproxen

    207.2 206.8 208.1 0.186 0.182 0.194 0.187

    Flurbiprofen

    220.2 219.8 221.1 0.382 0.37 7 0.391 0.383
    Sulindac 251.0 250.6 252.4 1.05 1.03 1.07 1.05
  3. Lab Practical

    Through a faculty mentor or collaborator, you should arrange to receive an unknown solution containing some combination of flurbiprofen, naproxen, sulindac, and tolmetin diluted in BGE at a concentration above the limit of quantification for your CE 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.