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High pressure liquid chromatography, or HPLC, is used to identify different solutes by changing their concentration in several mixtures in order to find the elution order. HPLC uses ultraviolet detection to identify and separate each solute. The elution order is based off polarity, size, and the molecular weight of the solute. The concentration of an unknown solution can then be found from a calibration curve based on the several mixture’s area ratio vs their concentration. HPLC can also be used to purify substances such as water or to detect impurities.
High pressure liquid chromatography is quicker than other methods and is a more affordable method.
In HPLC, there are two phases, a mobile phase and a stationary phase. The polar mobile phase is aqueous while the stationary phase is organic and non-polar. The solute can only move through the mobile phase in order to pass through the chromatogram and be separated by polarity or size. The time it takes for the eluate, or fluid emerging from the column, to pass through the column is the retention time.
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Different peaks are formed when the solute is separated. The order of solutes that leave the column is the elution order.
One problem with high performance liquid chromatography is the general elution problem. This problem happens when the retention time is too long to separate each peak to have a resolution factor that is greater than 1.5. To fix this, the organic phase is increased to the amount of the aqueous phase during the separation.
As a result, the mobile phase becomes less polar and the eluant strength decreases.
While HPLC can be used to purify substances, it can also be used in environmental situations as well.4 If a substance is questioned to be too toxic for the environment it is in, HPLC can help identify the toxicity of its components and help decide if that substance is a threat.
Table 1: Composition of Pharmaceutical Solutions
| Volume added (uL) | Solution 1 | Solution 2 | Solution 3 | Solution 4 | Solution 5 |
|---|---|---|---|---|---|
| Amitriptyline | 40 | 50 | 60 | 70 | 80 |
| Triprolidine | 50 | 60 | 70 | 80 | 40 |
| Promethazine | 60 | 70 | 80 | 40 | 50 |
| Pheniramine | 70 | 80 | 40 | 50 | 60 |
| Pyrilamine | 80 | 40 | 50 | 60 | 70 |
| Hexanophenone | 90 | 90 | 90 | 90 | 90 |
| Uracil | 50 | 50 | 50 | 50 | 50 |
Table 2: Chromatogram Data for the Standard
| Peak # | Retention Time (min) | Type | Width (min) | Area (mAU*s) | Height (mAU) | Area % |
|---|---|---|---|---|---|---|
| 1-Uracil | 0.405 | MM | 0.0340 | 752.28601 | 369.23743 | 10.8649 |
| 2-Pheniramine | 1.426 | BB | 0.0392 | 299.09766 | 119.61874 | 4.3197 |
| 3-Pyrilamine | 1.877 | BB | 0.0270 | 1158.91650 | 660.95789 | 16.7377 |
| 4-Triprolidine | 2.182 | BB | 0.0294 | 1801.37988 | 960.11121 | 26.0166 |
| 5-Promethazine | 2.831 | MM | 0.0268 | 859.66833 | 533.75970 | 12.4158 |
| 6-Amitriptyline | 2.966 | BB | 0.0274 | 1674.31335 | 958.16333 | 24.1814 |
| 7-Hexanophenone | 3.713 | MM | 0.0204 | 378.31021 | 308.74054 | 5.4638 |
Table 3: Chromatogram Data for the Unknown Sample
| Peak # | Retention Time (min) | Type | Width (min) | Area (mAU*s) | Height (mAU) | Area % |
|---|---|---|---|---|---|---|
| 1-Uracil | 0.408 | MM | 0.0342 | 446.45477 | 217.60190 | 10.4776 |
| 2-Pheniramine | 1.437 | BB | 0.0342 | 166.26254 | 75.43835 | 3.9019 |
| 3-Pyrilamine | 1.881 | BB | 0.0262 | 711.23297 | 420.66565 | 16.6925 |
| 4-Triprolidine | 2.190 | BB | 0.0267 | 747.25366 | 432.49481 | 17.5368 |
| 5-Promethazine | 2.834 | BV | 0.0249 | 788.41168 | 495.64539 | 18.5027 |
| 6-Amitriptyline | 2.972 | BB | 0.0256 | 1059.41516 | 647.33038 | 24.8627 |
| 7-Hexanophenone | 3.712 | MM | 0.0214 | 342.02563 | 266.47809 | 8.0268 |
Table 4: Average Concentrations of Each Solute in 3 Unknown Solutions (M)
| Solute | Concentration (M) |
|---|---|
| Antihistamine | 0.004 (± 0.5) |
| Promethazine | 0.005 (± 0.5) |
| Triprolidine | 0.006 (± 0.5) |
| Pyrilamine | 0.005 (± 0.6) |
| Pheniramine | 0.007 (± 0.7) |
Table 5: Separation Efficiency of the Standard from the Chromatogram
| Solute | Peak Capacity (n’c) | Peak Capacity (nc) | Plate Count (N) | Resolution (Rs) | Elution Factor (k’n) |
|---|---|---|---|---|---|
| Antihistamine | 137.4 | 43.27 | 64915.9 | 2.93 | 6.32 |
| Promethazine | 27.39 | 8.89 | 61818.7 | 13.59 | 5.99 |
| Triprolidine | 30.14 | 11.57 | 30515.8 | 6.36 | 4.39 |
| Pyrilamine | 18.54 | 7.83 | 26773.8 | 8.01 | 3.63 |
| Pheniramine | 3.29 | 1.64 | 7331.2 | 16.41 | 2.52 |
The elution order that was found was Uracil, Pheniramine, Pyrilamine, Triprolidine, Promethazine, Amitriptyline, and then Hyalophane. The resolution between these peaks were all found to be greater than 1.5, which meant that all the peaks were distinguishable from each other. This meant all the peaks were completely separated after they were sent through the chromatogram. Equation (3) found the plate count (N) for the solutes in table 4. The high numbers meant more peaks could be separated and the column was efficient. The k’n value that was found by equation (7) was 8.12 which was high. A high k’n value meant that not much of the sample was lost in the column. The k’n value was used in equation (6) which found the peak capacity for the gradient elution.
Although the calculated values for plate count, resolution, and k’n were all accurate, the concentrations found for each solute in solution in table 1 was not accurate. The R2 values in graphs 1-4 show that the concentrations found for the standard solutions were not accurate. This is due to the systematic error from pipetting. If the exact amount of each solute was not picked up in the pipette, the concentrations would vary greatly. To help fix this error, the solution should be inverted a minimum of 6 times to make sure there is a constant concentration throughout the solution.
Gradient elution is used when a sample varies in polarities. Using this type of elution allows for a higher resolution between peaks in a quicker amount of time. Although it is more timely, gradient elution requires more expensive equipment to undergo the process. Even with the complex equipment, it is still possible to fail to obtain a constant flow rate due to changes in the mobile phase which creates results that are hard to reproduce between samples.3 If the solute is not shaken before being inserted into the chromatogram, then it is possible the concentrations read will not give an accurate peak area.
Isocratic elution, comparatively, was best used when the mobile phase of the solution remained constant. Although isocratic elution was slower than gradient elution if the polarities varied, it was more likely to pick up changes in the flow rate and produces more accurate results if the rate was not constant. In the equation for isocratic elution (5), the peak capacity, or the number of peaks that can be separated, was much lower than the equation for gradient elution (6). The lower number of peaks meant it was less reproducible than gradient elution that had a higher peak capacity.
The purpose of this experiment was to identify the elution order of five pharmaceutical solutes and use their respective calibration curves to find the concentration of an unknown solute. The elution order found was pheniramine, pyrilamine, triprolidine, promethazine, amitriptyline and then hexanophenone. All the peaks were distinguished from each other in the chromatogram and based on the peak capacity for both gradient and isocratic elution, more peaks could be separated. An extension for this lab could be to use beer’s law to obtain more accurate results and compare those results to the original that were obtained using the calibration curve.
High-Pressure Liquid Chromatography (HPLC) for Solute Identification and Purification. (2024, Feb 18). Retrieved from https://studymoose.com/document/high-pressure-liquid-chromatography-hplc-for-solute-identification-and-purification
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