The sample in all separation experiments was a 30-mer oligonucleotide-A. The crude oligonucleotide-A obtained from IDT (USA) had a molecular weight of 9513. Reagent grade 1 M Tris HCl, acetonitrile, phosphate-buffered saline (PBS), and NaCl were supplied by VWR (USA). The on-site MilliQ system provided deionized water for the research (NANOPure). The chromatography stationary phase for the initial research was a Dionex DNAPac PA200 oligonucleotide column (4 × 250 mm). Before purification, all samples were examined on an Agilent 1260 analytical HPLC system. A 500 nmol of stock oligonucleotide-A was diluted to 1 mM in 10× PBS. To attain incoming purity, one nanomole of oligonucleotide-A was run on the PA200 column. After running the sample through the HPLC system, a mass was recorded using an Agilent 6100 series SQ system to confirm the product. The initial parameters in Table 1 show the analysis method used to record initial purity. The purity was documented, and the sample was readied for purification.

The preparative chromatographic studies were carried out on a ChemStation Work Station that was linked to an Agilent 1260 semi-prep HPLC system equipped with a diode array detector. Table 2 shows the experimental parameters that are being modified and tested. Each variant change was tested three times, with the given result being the average of the three runs. The first stage in the optimization process was to replicate the results provided in the literature, which showed that HPLC employing potassium phosphate buffer at pH 7 and acetonitrile yielded 88.0% purity and 80.00% recovery [1]. For peaks greater than 300 mAU, a shallow gradient was applied to an XTERRA C18 column (19 × 100 mm), and fraction collection was utilized.

Note: Each variant was run three times, the average of the three is reported in this work.

In-process samples from potassium phosphate buffer preparative runs at pH 5 to 9 were kept in the refrigerator at 4˚C. After that, the samples were concentrated using a Heidolph Precision rotary evaporator. The bath temperature was set at 30˚C, and the rotation speed was set to 100 rpm. The pressure was manually controlled, and it took roughly 75 minutes for one sample to evaporate from 40 mL to 2 mL.

A FreeZone Freeze Dry System was used to lyophilize the material. The leftover sample is frozen using dry ice and isopropanol alcohol after each sample has been evaporated close to dryness, 2 mL. This procedure takes approximately 5 minutes. The sample is placed on the Freeze Dry System overnight when it has totally frozen. The material was returned to powder form the following morning. After that, the sample is reconstituted in 10× PBS buffer.

After the material has been restored to liquid form, it is quantified. The quantification is done using a Biotek Synergy H1 spectrophotometer with the extinction coefficient of the oligonucleotide-A provided by IDT. The coefficient of extinction was 363,400∙M⁻¹∙cm⁻¹. The following calculation was used to determine concentration:

Concentration in uM = ( ( Absorbance * Dilution Factor Extinction . Coefficient ) / t ) * 1, 000 , 000

The final step in the procedure is reanalysis on an Agilent 1260 analytical HPLC system and verification on an Agilent 6100 Series SQ LC/MS system.

Table 3 lists all % purity values and yields with different pH values (5 to 9) at constant 10 mM K₂HPO₄, 20 mM K₂HPO₄, and 40 mM TEAB. Figure 1(a) compares the percent purity and yield of K₂HPO₄ at 10 mM, Figure 1(b) for K₂HPO₄ at 20 mM, and Figure 1(c) for TEAB at 40 mM, with pH values ranging from 5 to 9.

When the concentration of K₂HPO₄ was kept constant at 10 mM and pH 7, the purity was 87.5% and the yield was 79.0%, which stayed at the optimum level. However, lowering the pH results in a decrease in purity and yield. The purity of the sample was 86.0% at pH 6 and 85.9% at pH 5, with yields of 78.8% and 78.0%. This value is 1% - 2% lower than pH 7. The purity was 86.9% and 87.0%, respectively, and the yield was 79.1% and 78.9% at higher pH 8 and 9 but did not produce a greater purity and yield at this pH.

Note: Each value is obtained based on the average of three runs.

Changing the concentration of K₂HPO₄ from 10 to 20 mM and then varying the pH from 5 to 9 resulted in no improvement in oligonucleotide-A purification at a constant 20 mM concentration of K₂HPO₄. The pH 7 sustained optimum values reveal the same purity and yield as 10mM in the previous method as 87.7% and 77.9%, respectively. The overall purity at pH 6 and 5 was 86.7% and 86.3%, respectively, in the experiment, although the overall yield was 77.9% and 78.1%.

Yield was shown to decrease by 1.6 percent when the pH climbed from pH 5 to pH 9. The purity of oligonucleotide-A increased by 0.2 percent at 20 mM concentration and pH 7 compared to 10 mM concentration and pH 7. Yield, on the other hand, fell by 0.5 percent.

When the dipotassium phosphate buffer pH variation was compared, the results from the 20 mM concentration studies were all slightly higher than the results from the 10 mM concentration experiments. There was a 0.2% - 0.7% higher in purity at 20 mM K₂HPO₄ across pH 5 - pH 9. However, increased K₂HPO₄ concentrations resulted in a 0.2% - 1.4% decline in output.

When the buffer was changed from K₂HPO₄ to TEAB at 40 mM, the pH had a significant impact on the results. The purity and yield (93.3% and 74.5%, respectively) at pH 8 were equivalent to pH 9 (93.15% and 74.1%) at the same concentration; however, when compared to K₂HPO₄ at 10 mM, the purity increased by 6.1% while the yield decreased by 4.8%. When compared to K₂HPO₄ at 20 mM, the purity increased for TEAB at pH 8 and 9, but the yield was somewhat reduced.

When pH 5 and 6 were used with TEAB, the yields (93.0% for pH 5 and 93.7% for pH 6) were significantly higher (7.1 to 7.8%) than when pH 5 and 6 were used with K₂HPO₄. However, at the same pH levels, the yields were roughly 2.0% lower than with K₂HPO₄. TEAB at 40 mM and pH 7 produced the highest purity (93.9%); nonetheless, the percent yield is comparable to other pH values of the same buffer.

The standard deviation was calculated in these tests to quantify the amount of variation across all three trials at each pH level. All results were considered low, with the maximum standard deviation in the data set being 0.36 at pH 8 with 10 mM K₂HPO₄. The data set’s lowest standard deviation was 0.10, which was observed at pH 8 and pH 9.

According to the data in Table 3, at pH 7, both buffers (K₂HPO₄ and TEAB) produce the best outcomes when compared to the same concentration at other pH levels. This section explains the findings obtained when the buffer concentration varies from 1 to 40 mM while maintaining a constant pH of 7 (see Table 4 and Figure 2). At 1 mM concentration, K₂HPO₄ produced higher purity than TEAB (90.8% vs. 86.1%). TEAB, on the other hand, outperformed K₂HPO₄ by 5.4% in yield. At 10 mM concentration, TEAB produced 5.1% more purity than K₂HPO₄; nevertheless, TEAB produced 4.1% less yield than K₂HPO₄. Buffer K₂HPO₄ had around 5.1% less purity than TEAB at 20 mM concentration but had 2.8% higher yield. At 30 mM, TEAB has 6.0% higher purity than K₂HPO₄ and just 0.6% lower yield. Buffer K₂HPO₄ had 5.7% less purity than TEAB and 1.6% higher yield than TEAB at 40 mM. There were no significant variations in

Note: Each value is obtained based on the average of three runs.

purity or yield during pH tuning. However, increasing the concentration of K₂HPO₄ and TEAB had a greater impact on purity and yield. The drop-in yield during the pH experiment was not as extreme as the concentration decrease, but the purity increased dramatically.

The standard deviation was calculated based on all three experiments at each concentration level to measure the degree of variation. All concentrations were considered modest, although the data set’s biggest standard deviation was at 1 mM K₂HPO₄. With a standard deviation of 0.96, this experiment had the largest standard deviation of all the experiments. The data set’s lowest standard deviation was 0.10, which was observed at 1 mM and 10 mM for TEAB.

To optimize oligonucleotide-A purification, two additional buffers, TEAA and NH₄CH₃CO₂, were used in this study, and the experiment was performed at a constant pH of 7 and a constant concentration of 10 mM. The most change was generated by changing the nature of the buffer. Table 5 and Figure 3 show that TEAB had the highest purity at 92.6% (yield, 74.9%), followed by TEAA at 90.6% (yield, 72.1%), K₂HPO₄ at 87.5% (yield, 79.0%), and NH₄CH₃CO₂ at 84.3% (yield, 69.9%). According to the published literature [1], 88.0% purity and 80.0% yield were obtained by HPLC using K₂HPO₄ buffer at pH 7 (10 mM), while in this experiment, K₂HPO₄ purity was 87.5% and yield was 79.0%. There was a 5 -

Note: Each value is obtained based on the average of three runs.

6 percent higher from the earlier reported approach when TEAB was used instead of K₂HPO₄, and a 2.6 percent increase when TEAA was used instead of potassium phosphate. In all situations, the yield was reduced. The use of NH₄CH₃CO₂ reduced yield by 10.1%. TEAA reduced production by 7.9%, TEAB by 5.1%, and potassium phosphate by 1%. TEAB has the highest purity and yield of the four buffers tested; the purity is 5.4% greater than previously reported data, but the yield is lower. TEAB has the largest concentration, followed by TEAA and NH₄CH₃CO₂.

The standard deviation for the mobile phase experiment change was recorded, and the findings are shown in Table 5. The data set’s largest standard deviation was 0.66 at 10 mM TEAA. The data set’s lowest standard deviation was 0.10, which was seen at 10 mM TEAB buffer.

With purity improved from 88 to 94 percent, further investigation into expanding the study to different oligonucleotide-A chain lengths was pursued. Chain lengths ranged from 20 to 40 oligonucleotide-A. The same optimized procedure was used to treat these chains with TEAB at a constant pH of 7 and a concentration of 40 mM. Table 6 and Figure 4 summarize the outcomes of these experiments. In

Note: Each value is obtained based on the average of three runs.

chains longer than or equivalent to 30 oligonucleotide-A, the approach proved sufficient. Purity was found to be larger than the figure stated in the literature. The optimized process achieved a purity of 94% and a yield of 75%.