Capillary Zone Electrophoresis Experiment

Capillary Zone Electrophoresis

Introduction

 For separation of analytes from a matrix, the most common choice for analytical chemist is chromatography. Chromatography technique is good tools especially it able to deal with on almost mixture of analytes and matrices. However, on the emergence of complex problems that require more specific method, capillary electrophoresis come to overcome the problem. Unlike chromatography which the separation depends mostly on the interaction between analyte with mobile and stationary phase, capillary electrophoresis able to make separation, identify and quantify the analytes by differential migration of solute in an electric field ². This able to do a good separation for a matrix with combination of different m/z ratio. Capillary electrophoresis is the go-to technique for DNA sequencing due to its great speed².

 Capillary electrophoresis has a very simple instrument set up¹. The capillary with inner diameter around 25 to 75 μm is filled with buffer inside¹. At the both ends of the capillary are electrode with voltage control connected. In this experiment, the inner surface of capillary used is made up from silanol (Si-OH)¹. The key feature of how capillary electrophoresis instrument work is from the electroosmotic flow and electrophoretic mobility¹. Both parameters contribute to the total migration of the analytes in the capillary. Electroosmotic flow is due to buffer while electrophoretic mobility is due to the analyte.

The first step in capillary electrophoresis is to fill the capillary with a chosen buffer. Double layer will form a the silanol ionized and become negative charge and attract the counter ions from the buffer¹. Then, when a voltage is applied at both electrodes end, the positive charges at double layer are attracted to the cathode which created the electroosmotic flow¹. The buffer also dragged the analyte ions or molecules toward the cathode¹. The migration speed different between analytes depends on their charge and mass. Positive ion will attract to the cathode, while negative ion will do the opposite. The advantages of electrophoresis are high efficiency, low sample volume and wide applicability¹. However, the small variations in pH can make an impact on the result for using CE especially if there is peak that not able to make a good resolution with the second peak.

 In this experiment, there is 2 modules. The module 1 is to find the effect of pH on electroosmotic flow (EOF). It is deduced that the magnitude of EOF is proportional to the ionization of silanol surface. Therefore, three buffers with different pH 5.0, 7.0 and 9.5 are used for determining the DMFA migration time¹. The different on migration time can tell the effect of the pH of EOF. For the module 2, the objective is to determine the quantity of amino acid in Cutler Amino with standard addition¹. Amino acid is derivatized with chromophore, OPA-BME to make it detectable under UV-Vis detector. OPA-BME is used because the molecule is aromatic and form a bond with alpha amino¹. 

Experimental

Module 1: Preparation of buffer solutions, NaOH and DMFA.

The solutions of NaOH (0.1 M) was obtained from the fridge and 5 mL of the solution was transferred into a 10 mL beaker. The solution was filtered with a syringe filter into a new vial. 1 mL of the solution was transferred into a Beckman plastic molded vials and gray rubber caps with a designated micropipette. These steps were repeated for 0.1% DMFA, pH 5.0, 7.0, and 9.5 buffers.

Module 2: Preparation of Cutler Amino Pump.

The Cutler Amino Pump sample (0.15-0.20 g) was weighed with electronic balance and transferred into 50 mL volumetric flask. Fluorescein sodium salt (0.005 g) was added into the flask. Then, pH 9.5 borate buffers (25 mL) was added carefully into the flask. The flask was swirled carefully. 10 drops of NaOH (1 M) was transferred into the flask using a plastic pipet and the flask was swirled. pH 9.5 buffer was continuing to add to dilution mark and stir bar was added. The solution was stirred at medium speed until solution is dissolved. The solution was filtered with a syringe filter into another a 20 mL glass scintillation vial. This solution was kept in a drawer for Day 2 usage.

Module 2: Preparation of sample and standards.

The solutions of OPA-BME (0.03 M, 10 mL) was obtained from the fridge and was transferred into a glass vial. The solution was filtered with a syringe filter into a new vial. These steps were repeated for NaOH (0,1 M), pH 9.5 buffer, Asp spike solution (40mM), Leu spike solution (40 mM), mixed Asp/Leu spike solution (20 mM) for 5 mL each. The entire set of vials were degassed at once in the ultrasonic bath.

Conditioning the Capillary

On Day 1, the required solutions, 0.1 % DMFA, NaOH (0.1 M), pH 5.0, 7.0, and 9.5 buffers were put into a tray based on the lab manual instructions. The instructions from the lab manual were followed to set up the instrument for conditioning. For the first analysis run, the capillary was conditioned with pH 5.0 buffer. The method loaded set to run NaOH and the then pH buffer through the capillary. Refill the buffer into the vials back after conditioned. These steps were repeated for pH 7.0 and 9.5 buffers. On Day 2, the capillary was conditioned with pH 9.5 borate buffer only.

Analysis of samples

On Day 1, DMFA solution was analyze for different pH value at pH 5.0, 7.0 and 9.5 buffers. The overlay peaks at for each pH was printed. On Day 2, the Cutler only sample was run first. Then, Cutler + Leu, Cutler + Asp, Cutler + AA spike 1, Cutler + AA spike 2, Cutler + AA spike 3. Then, the peak area of Leu, Asp and fluorescein were determined after able to identify the peaks. The samples were analyzed and recorded at the University of Arizona on Beckman Coulter PA 800 for capillary electrophoresis with 32 Karat software program.

Results and discussion

For the module 1, the elution times of DMFA in buffer pH 5.0 and 7.0 were obtained as shown in Figure 1. The left peak was DMFA in buffer pH 7.0 while the broader right peak was DMFA in buffer pH 5.0. The module 1 also require obtaining elution times of DMFA in pH 9.5. However, due to not enough time, my lab partner and I unable to obtain the required data.

Figure 1. Elution times of DMFA in buffer pH 5.0 and 7.0.

Table 1. Table of elution times for DMFA in buffer pH 5.0 and 7.0.

The elution times of DMFA in buffer pH 7.0 was shorter in comparison to the DMFA in buffer pH 5.0. Therefore, it can conclude that as pH increases, the elution times become shorter. It can be predicted that elution time for DMFA in buffer pH 9.5 must be lower than 4.6 minutes. DMFA does not affected by the pH since it is a neutral compound, there the only factor changes the elution time is the electroosmotic flow. The result from this experiment shown that the properties of the analyte is crucial to determine the performance and factor effecting electrophoresis. It was concluded for faster elution of analyte higher pH is desirable.

Table 2. Raw data for samples volume and added concentration.

The effective added concentration of leucine and aspartic acid can be calculated using the formula, C_stdV_std/V₀.

Example calculation:

$\mathit{Effective\; concentation\; added\; for\; Cutler}+\mathit{AA\; micture }3 = \frac{400\mathit{\mu L}*20\mathit{ mM }}{2400\mathit{ \mu L }}$

$\mathit{Effective\; concentation\; added\; for\; Cutler}+\mathit{AA\; micture }3 = 3.33\mathit{ mM}$

Table 3. Leu, Asp and Fluorescein peaks area and ratio data for Cutler Amino samples.

In the lab, the leucine and aspartic acid peaks were determined by making comparison for electropherograms of Cutler, Cutler + Leu, and Cutler + Asp. This was done by overlay the electropherograms of all three samples. It was determined that leucine that leucine was the third peaks and aspartic acid was the eighth peak. Then, the peak area of leucine and aspartic acid were calculated for each sample. From the electropherograms, it clearly showed the advantages of electrophoresis by being able to produce a well resolved separations between peaks.

 Fluorescein was used for internal standard to eliminate any variation of factor that cannot be control such as the volume of injection by the instrument. Therefore, the peak area of internal standard at constant concentration was recorded. Then, ratio of peak leucine: fluorescein and aspartic acid: fluorescein was determined for each sample.

From the data in Table 2 and 3, calibration plot for leucine and aspartic acid can be developed with parameters of effective added concentration and ratio of peak area. Then, the concentration of the leucine and aspartic acid from the unknown can be determined by using the function of calibration plot and set the Y = 0 for initial concentration.

Figure 2. Calibration plot of peak area ratio against Leu concentration added.

Figure 3. Calibration plot of peak area ratio against Asp concentration added.

Example calculation:

$y=5.257x+12.25$

$\frac{y–12.25}{5.257}=x$

$x=\frac{–12.25}{5.257}$

Concentration of aspartic acid in Cutler sample = 2.33 mM.

$2.33\mathit{ mM }\times 0.05\mathit{ L}=1.17\times {10}^{–4}\mathit{mol\; Asp}$

$1.17\times {10}^{–4}\mathit{ mol\; Asp }\times 133.11\frac{g}{\mathit{mol}}=0.016\mathit{g\; Asp }\times \frac{1}{0.1828\mathit{ g\; Cutler}}$

Amount of Asp per gram of cutler = 0.085 g Asp / g Cutler.

Table 4. Amount of Leu and Asp added in Cutler Amino samples in moles and gram.

From the calculation above and data in Table 4 it was determined that the initial concentration of leucine and aspartic acid in the Cutler solution were 0.55 mM for leucine and 2.33 mM for aspartic acid. From here, a further calculation was done to determine the mass of leucine and aspartic acid from the mass of Cutler obtained, 0.1828 g. The mass of leucine and aspartic acid per gram of cutler was 0.020 and 0.085.

The experiment result obtained was good. This can be explained from a well resolved peaks, point on calibration plot within the range and usage for internal standard to eliminate variations of uncontrollable factors. This experiment could be improved by taking more extra precautions when carrying out the experiment to avoid human error and mistake. Time was not enough to complete the module 1 due to mistake during capillary conditioning. To reduce error in the final values and ensure for precision and accuracy, multiple run for the sample could be done. Increase number of spikes also can produce a very accurate result.

Questions

1.1 In module 1, to determine only the electroosmotic flow of CZE at any pH value, any other factor should be eliminated, this including the contribution of electrophoretic mobility from charge molecules. Neutral marker was used because the electrophoretic mobility is none and therefore no influence of electrostatic force from the electrode if pH value changes. If the marker compound change in response to the environment, the signal detected also will change. DMFA is not ionizable, because the net charge for both electronic structures is 0.

1.2 From the trend of migration that can be observed in Figure 1, the elution times for DMFA decrease as the pH value is increases. As the pKa of silanol group is within ~3.5 to 4.5, when the pH of the condition increases, more silanol group become ionizable. Hence, more counter ions attracted to the Si-O^– to form a double layer. In comparison to pH 9.5, pH 5.0 is not basic enough to effectively form a double layer for EOF. Therefore, when voltage is applied, the amount of counter ions from double layer attracted to the cathode at higher pH is more than at low pH value. This effectively drag the analyte toward the cathode end and the elution time become shorter.

1.3 As both pH 7.0 and 9.5 are above the pH 6, most of silanol group already ionize at both conditions. Therefore, for completely ionize group the % ionization at both pH should be almost 99%. As the pH keep continue to increase the % ionization become plateau. At pH 9.5 there is excess in cations remain in the solution which give slight change in EOF and migration time in comparison to pH 7.0.

2.1 At a very basic pH, amino acid with positive side chain will probably be neutral if the side chain pKa value lower than the pH value. If higher it will be positive. For neutral side chain of amino acid, it will be a neutral and for amino acid with negative side chain it will be negative. At pH 9.5, alpha amino is no protonated, so leucine has negative, alanine has is negative, and aspartic acid is doubly negative.

2.2 Charge is the main factor on determining the elution order of compounds. At pH 9.5, aspartic acid with charge of double negative should elute first compare to alanine and leucine with charge of single negative. Compound with more negative charge will elute faster compare to more positive charge compound. Then, to determine the elution order of identical net charge size difference can become the next factor. Since alanine is smaller then leucine, it will elute before leucine.

2.3 The remaining OPA which did not react with the amino acid is a neutral species. Since OPA is a neutral species if there is amino acid with positive side chain, at pH 9.5 both peaks become closer together. This is because, the amino acid with positive side chain at pH 9.5 has a net charge of 0.

2.4 One serving has 9.33 g of Cutler Amino Pump. From the calculation and data from table 4, the amount of leucine and aspartic acid for one serving are 0.19 g and 0.79 g respectively. The total mass of amino acids is 0.98 g which is almost accurate just off on the upper side compare to 8.1 g. This proven the data obtained in this experiment reliable. 

References

[1] Belle-Oudry, Chem 400A Lab Manual, 2018, 85-102.

[2] Heiger, D., High Perfomance Capillary Electrophoresis: An Introduction, Agilent Technologies, 2000.

[3] Harris, D. C., Quantitative Chemical Analysis, 8^th ed., W. H. Freeman and Company, New York, 2010.

[4] Liu, J., Cobb, K. A., and Novotny, M., J. Chrom., 468, 1988, 55-65.

[5] Roth, M., Anal. Chem., 43, 1971, 880-882.

[6] Simons, S. S. Jr., and Johnson, D. F., J. Amer. Chem. Soc., 98, 1976, 7089-7099.