Biology论文模板 – High-Performance Liquid Chromatography (Hplc) Report

Summary

Separation and quantification of the oligonucleotides is a key step in biological engineering. The aim of this experiment was to separate oligonucleotides components in a mixture and quantify the concentration of the unknown solution of oligonucleotides using High-performance liquid chromatography (HPLC) data. The quantification step involved the generation of a calibration curve from the chromatogram peak area of the three different concentration of oligonucleotide A. The calibration curve equation was then used to determine the concentration of the unknown. The concentration of the unknown was determined to be 47.94. It was evident that HPLC provides an efficient quantitative separation of oligonucleotides.

Aim

The aim was to separate the oligonucleotide components in a mixture and quantify the concentration of the unknown solution of oligonucleotides using HPLC data.

Introduction

Oligonucleotides are important components in gene synthesis. The oligonucleotides are preferred for use in the synthesis of genes since they are made up of a relatively smaller number of nucleotides (Hughes et al., 2001). The process of gene synthesis involves the addition of several bases to form a continuous DNA strand. Short oligonucleotides are often used in biological engineering as primers to initiate the process of DNA replication. The oligonucleotide can also be used to synthesize DNA strands with longer sequences that can be used as genes (Wang et al., 2009). The assembly of genes of interest from short oligonucleotides involves the synthesis, identification separation and the quantification of the target oligonucleotides before ligating them together to form the gene of interest.

The separation of the oligonucleotides is a sensitive step, which requires the use of a highly efficient, specific and sensitive approach. The use of HPLC enables the separation of the oligonucleotides while ensuring that the configuration of the strands and function is not altered (Kawasaki et al., 2011). High-Performance Liquid Chromatography can also be used in the quantification of the separated oligonucleotides (Gilar et al., 2003). The modern HPLC device (Figure 1) is made up of a pump, an injector, column detector and a computer. The pump is used to force the sample introduced into the mobile phase by the injector through the column. The column separates the components of the mixture while the detector identifies and quantifies the separated analyte through chromatogram output in a computer (Swartz, 2005).

Figure 1: The modern HPLC device (Zotou, 2012)

The quantitative ability and the accuracy of HPLC in the separation of molecules have resulted in the wide application of the technology in other fields such as in the manufacture of drugs and other biological products and the detection of illicit drugs in urine (Castiglioni et al., 2006). The technique is also used in healthcare in the detections of target biomolecules in the blood (Meulenberg, 2012). In this experiment, target oligonucleotides were separated using HPLC. The unknown oligonucleotide was then quantified based on the calibration curve.

Experimental Methodology

The following steps were used in the sample analysis:

  • 5 μL of oligonucleotides A, B and C were pipetted to a provide sample vial and 485 μL added and vial labeled as oligonucleotide mix.
  • Oligonucleotide A concentrations of were then prepared as shown in the table below and the vials labeled appropriately
Sample concentration (μL/mL)Volume of oligonucleotide (μL)Volume of tris buffer (μL)Total Volume (uL)
12.525197.5200
255195200
5010190200
  •  All the prepared vials and the provided vial containing the unknown were placed in the tray of the autosampler and proper instructions on the positioning of the vials provided to the software.
  • First, the oligonucleotide mix vial was analyzed for 10 minutes and the chromatogram obtained. Vials with known concentrations of oligonucleotide A followed by a vial of the unknown were also analyzed in the same way and chromatograph obtained.
  •  The calibration curve was then created and used to determine the concentration of the unknown based on its peak.

Principle and Theory

The principle that was employed in the laboratory session is based on the separation of the oligonucleotides from the mixture facilitated by the difference in the retention times. Retention time refers to the time taken from injection of the sample to detection as evident by the chromatogram peak (Gika et al., 2007). The separation of the different components of the mixture was carried out by passing a mobile phase through a column containing stationery phase. The difference in the binding of the components to the stationery phase led to the elution of the components from the column at a different time. The components that bind tightly to the column take more time to be eluted (Singh and Mehta, 2006). This experiment was performed based on the assumption that the components of the mixtures had unique chemical and physical properties such as polarity, charge, and molecular weight. The samples were injected into the column, and the difference in the properties of the components led to the difference in column packing as the components of the mixture interacted with the components of the stationery phase (Gritti et al., 2010). The oligonucleotide was eluted individually at a specific time. Ultraviolet detectors set to 260 nm were used in this experiment since the oligonucleotides absorb UV at 260nm (Clavé et al., 2014). The eluted oligonucleotide was then detected by a detector and the signal output displayed as a chromatogram with specific peaks. The identification of the oligonucleotide was done based on the characteristic of the chromatogram peak which includes the area and the height. 

The determination of the concentration of the unknown in the experiment was done by the use of generated calibration curve of area versus concentration of oligonucleotide A. The calibration curve usually depicts a linear relationship as shown in figure 2.

Figure 2: The standard calibration curve

The peak area of the unknown was then used to determine the unknown concentration based on the calibration curve equation.

From Figure 2 the curve equation is in the form of y=mx+c where y is the area and x is the concentration. The concentration of the unknown was therefore determined by rearranging the curve equation and substituting for the area of the unknown oligonucleotide. 

Results and discussion

The HPLC analysis of oligonucleotide A of different concentrations and the unknown oligonucleotide yielded chromatograms of varied heights and area at UV wavelength of 260 nm as shown in Table 1. 

Table 1:  The data of area and height and retention time derived from the chromatograms

Concentration μL/mLAreaHeightRetention time (min)
000 
12.50.06381.096.7
250.4976.646.713
502.379224.916.695
X1.806220.826.703

      The peak area obtained from the chromatograph of the known concentrations of oligonucleotides A was used to generate a calibration curve shown in Figure 1.

Figure 3: The calibration curve of peak area of chromatograms of oligonucleotide A against the concentration

The concentration of the unknown oligonucleotide was then calculated based on the equation of the calibration curve as follows:

From the equation of the Figure 3 curve, y= 0.0403x, where y and x is the peak area and concentration of the unknown oligonucleotide.

By rearranging the equation of the calibration curve in Figure 3:

Concentration (x)= Peak area (y)/ gradient (0.0403)

Based on the values of peak area in Table 1:

x= 1.8062/0.0403= 44.819 μL /mL

Therefore, the concentration of the unknown oligonucleotide is 44.819 μL /mL. The results obtained on the concentration falls within the expected range based on the results on the height and area of the various concentration of oligonucleotide A samples. The peak height of oligonucleotide A at a concentration of 25 μL /mL was 6.64 while at concentration 50 the peak height was established to be 24.91 μL /mL. The concentration of the unknown therefore lies between the predicted value of 50 μL /mL and 25 μL /mL as indicated by its peak of height 20. 82. These results support the suggestion that the size of the peak, both in terms of area and height, should be proportional to the concentration of the substance being analyzed (Marini et al., 2011). The use of peak area in the determination of the concentration of the unknown is based on the fact the peak area is less affected by the speed at which the analyte moves through the detector (Hutchinson et al., 2011).

The experiment, has therefore, shown that HPLC can be used to separate and determine the concentration of the unknown analyte. The results obtained in this experiment could have been affected by errors since the coefficient of R obtained from the curve was below 0.996. Some of the errors that might have occurred include the measurement errors and detector non-linearity.

Conclusion

The aim of the experiment, which was to separate and quantify the unknown oligonucleotide was achieved. The concentration of the unknown was determined to be 47.94 μL /mL. Knowledge on how to operate the HPLC device and to interpret its output was also gained. An understanding of the steps involved in the preparation of the calibration solutions and the how to generate and use calibration curve was also gained. It was evident from the experiment that HPLC is a quick and efficient method for separation of a component from a mixture. The method is, therefore, appropriate for use in quantitative separation of oligonucleotides used in biological engineering and industries. 

References

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Clavé, G., Chatelain, G., Filoramo, A., Gasparutto, D., Saint-Pierre, C., Le Cam, E., Piétrement, O., Guérineau, V. and Campidelli, S., 2014. Synthesis of a multibranched porphyrin–oligonucleotide scaffold for the construction of DNA-based nano-architectures. Organic & biomolecular chemistry, 12(17), pp.2778-2783.

Gika, H.G., Theodoridis, G.A., Wingate, J.E. and Wilson, I.D., 2007. Within-day reproducibility of an HPLC-MS-based method for metabonomic analysis: application to human urine. Journal of proteome research, 6(8), pp.3291-3303.

Gilar, M., Fountain, K.J., Budman, Y., Holyoke, J.L., Davoudi, H. and Gebler, J.C., 2003. Characterization of therapeutic oligonucleotides using liquid chromatography with on-line mass spectrometry detection. Oligonucleotides, 13(4), pp.229-243.

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