Chromatography Experiment

1. Purpose of the experiment

The purpose of the experiment is to understand the working principle of chromatographic separation, which is the separation of solids in a mixture by subjecting the solids to flow using a mobile liquid through a stationary stable phase. Hence, the solids tend to separate based on their interactions with the stationary phase while they flow. Another reason for this experiment is to understand the principle and technique used to determine the inter-particle and intra-particle porosity of columns. Also, to separate a two-solid mixture using column chromatography, the selectivity of the mixture must be calculated based on isotherm parameters and porosities, and the solute movement theory must be understood. Lastly, to understand factors that affect the mass transfer of solutes, such as extra-column dead volume.

2. Equipment materials

Distilled deionized water (DDW) (Mobile solution)
Liquid pump
Blue dextran, sodium chloride (NaCl), tryptophan (TRP) and phenanthrene (PHE) as feed (Feed)
Feed injector
Polyvinyl pyrrolidone (PVP) resin (adsorbent) in a column
Detector

3. Experiment procedure

Use a liquid pump to dissolve DDW solvent into the sample of blue dextran carefully
Inject the solution using a feed injector into a column filled with PVP resin adsorbent
Connect the column to the detector
Observe and record data shown in the detector.
Repeat the procedure using NaCl, Trp and Phe solutes

4. Data/ observations

Given 
Length of the column (LC) = 23cm
S = 1.7671cm 2
Q = 3ml/min

5. Result Analysis

Extra column dead volume in the separation system
RT * flow rate = dead volume
Inter-particle and intra-particle porosities of column
Selectivity for the separation of phenanthrene and tryptophan
General formulae
Where tvoid =Vvoid/Q

6. Discussion

Chromatography is one of the most effective tools in separating substances in chemistry and biological and life science. The separation of amino acids is considered an effective approach in studies involving peptides and protein analysis. Aromatic amino acids such as tryptophan and phenylalanine cannot be separated easily using conventional separation methods. This is because their physical as well as chemical properties are almost the same and can definitely adhere to a similar matrix in separation protocol, thus limiting the efficiency of these methods. The use of liquid chromatography and high-performance liquid chromatography are considered as the golden for such studies. A liquid chromatography method based on reverse-phase separation is effective in the separation of this aromatic amino acid, such as tryptophan and phenylalanine, without providing modification on these amino acids (Hesse and Weller n.p). Reverse-phase chromatography is lucrative in these studies as it ensures that the peak formed will not overlap, thus interfering with the interpretation of the results obtained from the study.

After the preparation of the sample, it should be injected into an HPLC system and allowed to undergo the mobile phase for 30 minutes. The most efficient mobile phase or formulation of these amino acids should be carried out to ensure success is achieved. These solutions include acetonitrile and deionized water to avoid binding to the amino acids, thus disrupting the separation process. Calibration of the system in most cases is done using homotyrosine and 4-fluorophenylalanine.

Separation of these aromatic amino acids was made possible due to the different extinction coefficients of these amino acids in water as well as other mobile phases used in the set-up. The confirmation of the presence of these amino acids in most cases is done by checking for absorbance of the filtrates obtained at the end of the whole cycle (Hesse and Weller n.p). Aromatic amino acids have different absorbance, it is possible to differentiate tyrosine from tryptophan from other amino acids such as phenylalanine as at 280nm as only the two absorbs.

Separation of this aromatic amino acid is made possible due to the fact that these molecules had a different extent of polarity. The amino acid with the largest hydrophobic part was retained in the HPLC system; thus, it was eluted last. Isomers, in this case, cannot be separated using this approach. An additional technique must be involved for separation success to be realized. Various factors usually appear to influence the effectiveness of chromatographic techniques used to separate various molecules. Some of these factors, including column length, particle diameter flow rate, and particle size distribution, affect the efficiency of HPLC. Additionally, separation is also affected by temperatures, the stationary phase, and the composition of the eluent, among others.

When performing a chromatographic experiment, there are certain desired volumes which, once used, efficiency of the experiment is achieved. In most cases, reducing the extra volume when using smaller volume HPLC columns is necessary to ensure that the system is not overwhelmed, thus giving distorted results. Extra-column dead volume, in most cases, affects separation, as once injection is done into the system, the molecules to be separated are dispersed in all directions due to different concentration gradients at the outer edges of a particular band. This will make the band broaden interfering with the whole system tubing as well as mobility of molecules thus affecting the separation process.

Conclusion

High-performance liquid chromatography remains the most effective separation method for aromatic amino acids when analysis of these molecules is needed. Here, separation occurs based on the hydrophobic nature of the molecules being separated. In separating phenylalanine and tryptophan, the most hydrophobic amino acid is eluted first, and the least the most hydrophilic amino acid is retained for a longer time within the column. It is important to ensure that these substrates are adequately prepared to enhance separation. The purity of the eluant should be determined using absorbance under spectrophotometry at a specific absorbance. this, in particular, is important for other studies requiring these amino acids.

Works Cited

Hesse, Almut, and Michael G Weller. “Protein Quantification by Derivatization-Free High-Performance Liquid Chromatography of Aromatic Amino Acids.” Journal of amino acids vol. 2016 (2016): 7374316. doi:10.1155/2016/7374316