# 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.