# Procedures: The Module

1. How are the analytes separated by MEKC? Describe fundamental principles that govern the method. With MEKC, analytes are separated based on their affinity to a micellar pseudo-stationary phase added to the running buffer. Micelles are comprised of surfactant molecules that contain both hydrophobic and hydrophilic regions. When the surfactant concentration is greater than the critical micelle concentration (CMC), the surfactant molecules aggregate to form micelles. These micelles have an inherent velocity in a capillary electrophoresis separation based on the micelle charge-to-size-ratio, which dictates electrophoretic velocity as well as electroosmotic velocity. Analyte that partitions or interacts with the micelle will assume a migration velocity identical to that of the micelle while the analyte interacts (or is associated) with the micelle. When the analyte dissociates from the micelle, the analyte velocity is a function of the analyte charge-to-size-ratio (electrophoretic mobility), as well as electroosmosis. Therefore, the stronger the affinity between an analyte and micelle, the more time the analyte will spend associated with the micelle. The more time the analyte spends associated with the micelle, the more similar will be the migration time of the analyte and micelle. Two different analyte, analyte A of high micelle affinity and analyte B of low micelle affinity can be separated from each other using MEKC, even though they may be inseparable by free zone capillary electrophoresis. A series of analytes that cannot be separated using free zone capillary electrophoresis, but possess varying micelle affinity, can be separated by MEKC. There are many different surfactants which can be incorporated in MEKC as pseudo-stationary phases. Sodium dodecyl sulfate (SDS) is an anionic surfactant that is commonly used in MEKC.
2. Define retention factor (or capacity factor) as it pertains to MEKC. Write the equation you would use to calculate the retention factor of tolmetin in pH 10, CAPs buffer 100 mM SDS. Describe what experiments are necessary to obtain each factor. The retention factor is the ratio of moles of analyte in the micelle to moles of analyte in the aqueous running buffer. It is a quantitative measure of analyte-micelle affinity. The electroosmotic velocity, micelle velocity and analyte electrophoretic velocity in the micelle loaded running buffer are necessary to calculate this value. The electroosmotic velocity and micelle velocity are measured experimentally using a standard neutral marker and hydrophobic micelle marker such as n-decanophenone or Sudan III.
 $k' = \dfrac{t_{tolmetin\_MEKC}\left(1+\dfrac{\mu_{eph\_tolmetin}}{\mu_{eof\_MEKC}}\right) - t_{eof\_MEKC}}{t_{eof\_MEKC}\left(1 - \dfrac{t_{tolmetin\_MEKC}}{t_{n−dec\_MEKC}}\right)}$ 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-

To determine the five values listed above, you must conduct two separate experiments. The first experiment is a series of free-zone runs (n = 3) with neutral marker and the analyte (dimethylformamide and tolmetin, respectively). This experiment provides migration times which contribute to the calculation of μeph_tolmetin. The following values must be determined or measured: the applied separation voltage (Vsep), the length of the capillary from the anode to the detection window (Lw) in cm, the total length of the capillary (Lt) in cm, and migration times in seconds. The units for mobility are cm2/Vs. To calculate μeph_tolmetin use the following equation:

μapparent_tolmetin = μeph_tolmetin + μeof

μeph_tolmetin = [Lw x Lt]/(tanalyte x Vsep)

μeof = [Lw x Lt]/(tneutral_marker x Vsep)

The second experiment is a series of MEKC runs (n = 3) with a neutral marker, the analyte, and a hydrophobic marker (dimethylformamide, tolmetin, and n-decanophenone, respectively). The MEKC run buffer is same as the free-zone buffer with the exception that it also contains the surfactant (SDS). The second experiment provides the migration times used to calculate ttolmetin_MEKC, teof_MEKC, tn-dec_MEKC, and μeof_MEKC. To calculate μeof_MEKC use the following equation:

μeof_MEKC = [Lw x Lt]/(tneutral_marker(MEKC) x Vsep)

Once the five specified values and the three constants are determined, k’ can be calculated using the equation provided above. For the best precision, the k’ values should be calculated for each individual trial and the mean of these k’ values should be reported. For sample calculations, see question 4.

3. Write a standard operating procedure for the separation of an unknown sample using MEKC. Assume this “unknown” contains three NSAIDS dissolved in background electrolyte (BGE). Be sure to include instructions for making BGE and implementing the technique. The background electrolyte buffer consists of micelle-forming surfactant added to standard free-zone CE run buffer. The appropriate mass of surfactant is added to a clean, empty vessel. Next, the calculated amount of run buffer is added to the vessel to produce a background electrolyte solution with the desired surfactant concentration. The solution is then thoroughly mixed agitated until the surfactant is completely dissolved. The background electrolyte must be stored at room temperature and is best if used the same day it is made. The vials containing the background electrolyte for use in flushing and for the anodic and cathodic reservoirs must be degassed.

Once the unknown analyte sample is analyzed by MEKC, there are two methods for determining the identity of the unknown analyte. The first involves preparing a series of standards. Each standard contains a neutral marker, one NSAID, and a micelle marker. The neutral marker should be neutral and not particularly hydrophobic (dimethylformamide), while the micelle marker should be neutral and quite hydrophobic (n-decanophenone or Sudan III). The reference samples are subjected to the same separation parameters as the original unknown sample. If done properly, the migration times for the neutral marker and micelle marker should match the times obtained from in the unknown sample. If the analyte migration time from the reference sample matches the one from the unknown, the NSAID from the reference is the unknown analyte. A comparison of analyte migration times between all reference samples and the unknown sample should be examined to positively identify the analyte. If the migration times of the reference samples are sufficiently close enough to each other to introduce uncertainty in the identification of the unknown analyte, the capacity factor, k’, determined from replicate trials can be used to ascertain if that particular NSAID is present in the unknown sample. The second method for determining the identity of NSAIDs in a mixture of unknown NSAID composition involves spiking standard NSAIDs in the unknown NSAID mixture. With this method, the unknown sample is run first. Then aliquots from a series of NSAID standards are added sequentially to the unknown. If the added NSAID results in a new peak on the electropherogram of the unknown sample, then the added NSAID is not present in the unknown sample. If the added NSAID results in an increased peak height for the unknown analyte peak in the original electropherogram, then the added NSAID is the present in the sample. While the spiking method might be the quicker of the two methods, it is best suited for identifying the composition of a mixture comprised of a limited set of analyte and the user must have prior knowledge of the response for each NSAID at a particular concentration. The first method has the added benefit of resulting in a series of three-peak electropherograms as opposed to multiple-peak electropherograms resulting from the standard addition method.

4. Determine the migration time for dimethylformamide and tolmetin for 3 consecutive free zone capillary electrophoresis runs. In addition, determine the migration time for dimethylformamide, tolmetin and n-decanophenone for 3 consecutive MEKC runs. Sample should be dissolved in background electrolyte (BGE). The sample for free zone capillary electrophoresis should contain 0.01% DMF and 300 micromolar tolmetin, while the sample for MEKC should contain 0.01% DMF, 300 micromolar tolmetin, and 300 micromolar n-decanophenone. This run buffer, or BGE, for the free zone capillary electrophoresis is 25 mM CAPs buffered to pH 10. The run buffer for the MEKC separations is 25 mM CAPs, 25 mM SDS buffered to pH 10. You must use a ~25 micron inner diameter fused silica capillary ~42 cm total length, ~32 cm to the window, 20 kV applied voltage. Be sure to flush the capillary adequately before each set (either free zone or MEKC). You may use the tables below to organize your data.
Free Zone DATA
Lw 32.9, Lt 42.8, 20,000 V, pH 10 CAPs
Trial Migration Time DMF Migration Time Tolmetin μ eof (CE) tolmetin μ app tolmetin μ eph
1 91.4 119.9 0.0462 0.03522 -0.0110
2 91.9 120.6 0.0459 0.03502 -0.0110
3 94.4 123.3 0.0448 0.03427 -0.0105

AVERAGE tolmetin μ eph -0.0108

MEKC DATA
Lw 32.9, Lt 42.8, 20,000 V, pH 10 CAPs 100 mM SDS
Trial Migration Time DMF Migration Time Tolmetin Migration Time n-dec μ eof (MEKC) tolmetin k'
1 105.5 148.5 259.0 0.04004 0.0652

2

105.9 149.2 263.1 0.03988 0.0619
3 107.0 150.5 264.7 0.03948 0.0500

AVERAGE tolmetin k’ 0.0590