- Syringes – 25µl, 500µl,1000µl, 2000µl.5000µl
- Injection loops – 5ul, 10µl, 20µl, 100µl, 200µl, 500µl, 1000µl, 2000µl, 5000µl
PROCEDURE
COLUMN 1: 100 × 4.6mm column of 4µ Genesis 120Å C18
Set volume – concentration varied
- Firstly, the eluent was made up. This was 12% acetonitrile buffered to pH 4.7. A 50 mmol buffer was made by weighing 3.85g of Ammonium Acetate (M.W=77.08g) in 1000ml of water. Acetic acid was added dropwise using a pH meter and magnetic stirrer to pH 4.7. This mixture was then filtered under vacuum and used to make 12% Acetonitrile.
=880ml buffer solution to 120ml acetonitrile gives 1000ml of 12%
Acetonitrile.
- Standard solutions of Caffeine, Theophylline, sodium Benzoate in 10ml of 12% acetonitrile were made up:
40mgs Acetone
80mgs Theophylline
120mgs Caffeine
450mgs Sodium Benzoate
(All weighed to 4 d.p on an analytical balance)
Actual Weights:
1. Acetone = 0.0688g
2. Theophylline
- Weight of weighing boat and sample = 0.0830g
- Weight of weighing boat after sample removed = 0.0024g
- Weight of sample = 0.0806g
3. Caffeine
- Weight of weighing boat and sample = 0.1260g
- Weight of weighing boat after sample removed = 0.0008g
- Weight of sample = 0.1252g
4. Sodium Benzoate
- Weight of weighing boat and sample = 0.4576g
- Weight of weighing boat after sample removed = 0.0024g
- Weight of sample = 0.4550g
Concentrations:
1. Acetone
M.W 58.08g
Number of Moles = Mass = 0.0688 = 0.00118 moles in 10ml
M.W 58.1
0.00118 × 100 = 0.1184 mol dm-3
2. Theophylline
M.W 180.17g
Number of Moles = Mass = 0.0806 = 0.000447 moles in 10ml
M.W 180.2
0.000447 × 100 = 0.0447 mol dm-3
3. Caffeine
M.W 194.19g
Number of Moles = Mass = 0.1252 = 0.000645 moles in 10ml
M.W 194.2
0.000645 × 100 = 0.0645 moldm-3
4. Sodium Benzoate
M.W 144.11
Number of Moles = Mass = 0.4550 = 0.00316 moles in 10ml
M.W 144.1
0.00316 × 100 = 0.316 mol dm-3
- 1ml of each solution was taken to produce a standard solution. This was then diluted to give solutions of different concentrations:
1 ml was taken and made up to 10ml with water to give a 1 in 10 solution and so on to give the following dilutions-
1 in 10, 1in 100, 1 in 1000, 1in 10000 and dilutions in-between.
Flow rate: 1ml/min
Response: 0.5
Run-time: 10 mins
Wavelength: 254nm
Range: varied depending on the concentration (see chromatograms)
Volume: 5µl
- Computer method to start run:
- Select Acquire/Reprocess
- Select Acquire data
- Zero baseline
- Syringe filled ensuring no air bubbles
- Load sample
- Inject sample and trigger (alt 1)
- 5 – 10 mins were allowed between runs.
- Dilute solutions were injected first.
- Syringe was washed with acetonitrile after every sample used.
- The % efficiency was calculated from the number of plates/meter and concentration versus efficiency was plotted.
RESULTS
Discussion
The peak for acetone is very small and hardly distinguishable due to the large number of peaks around its retention time, including a strong negative peak, which could affect its efficiency. The acetone could have evaporated due to its high volatility. The presence of acetone is to give t=0 and hence to calculate k` values and so the peak can be ignored as it will not affect the other peaks and their efficiency. The retention time for this unretained peak is 1.43mins.
The % efficiency for all three compounds is calculated relative to the 1 in 10000 dilution, which is set at 100% efficiency. The numbers of plates per meter all have standard error reproducibility. This accounts for deviations at lower concentrations that could be expected to be constant until the column becomes overloaded and the efficiency decreases.
The efficiency of this column remains fairly steady between the1 in 10000 and 1 in 5 solutions for the three compounds. There is then a large significant decrease between the 1 in 5 and the standard solution. It could be suggested that this is the region of concentration in which the column becomes overloaded.
The symmetry values for the peaks at 10% height remain fairly constant for theophylline from the most dilute solution to the 1 in 5 solution but then decrease between the 1in 5 and standard solutions. This seems to mirror the results for efficiency. The efficiency decreases as the peaks are becoming more asymmetric and the column is overloading. Caffeine follows a similar trend but there also seems to be a decrease between the 1in 10 and 1 in5 solution too. The symmetry results for sodium benzoate on the other hand, does not follow the same sort of trend but fluctuate considerably. All symmetry values for the three compounds are less than 1, which indicate that b>a and suggest tailing of peaks.
B. Set concentration – volume varied
- Procedure as in A with the same HPLC conditions but the concentration was kept constant and the volume varied.
- A fairly dilute solution was used (1in 800) to ensure that by increasing the volume a concentration effect would not be seen.
2µl, 5µl, 10µl, 20µl, 50µl, 100µl, 200µl, 350µl, 500µl, 750µl, 1000µl, 3000µl
5µl, 10µl, 20µl, 100µl, 200µl, 500µl, 1000µl, 2000µl, 5000µl
Analomous results occurred when a small volume was injected onto a large loop. Each volume was injected onto a bigger loop to find out if part filling the loop had an effect on the efficiency of the peaks. (See results table)
- The % efficiency was calculated from the number of plates/meter and volume was plotted against efficiency. The most accurate values of the number of plates/m were converted into % efficiency and plotted.
RESULTS
Discussion
The peak for acetone is indistinguishable. Retention time for this unretained peak is 1.43 min.
Different size loops were used for each volume to discover if using a larger loop would affect the efficiency values. In principle, this should not be the case as it was ensured that the arrangement on the valve was such that the sample would go straight onto the column and not have to travel the length of the loop. However, the results show a general decrease in efficiency when a larger loop is used for a small volume. So, the most accurate values for plates/m were converted to % efficiency and plotted.
The % efficiency is calculated relative the 5µl volume as the 2µl value is low compared to the others. The efficiency for 2 and 5 µl injected onto a 5µl loop were lower than the other volumes suggesting perhaps the presence of a dead volume in the loop. This could occur if the ferrel is too short or too long. Thus, the efficiency values on the 5µl loop are regarded anomalous and are to be ignored.
The efficiency of the column with theophylline remains fairly steady between 2µl and 1000µl showing no sign of overloading.
For caffeine, the efficiency remains stable between 2µl and 200µl. There is then a ~10% decrease between 200µl and 350µl, followed by another decrease of ~4% between 350µl and 500µl and a ~2% decrease in efficiency between 500µl and 1000µl. This suggests that the column becomes overloaded with caffeine between 200µl and 350µl.
With sodium benzoate however, the efficiency remains steady between 2µl and 100µl, there is then a ~5% decrease between 100µl and 200µl, followed by ~17% decrease between 200 and 350µl and a ~13% decrease between 350µl and 750µl. The value for 1000µl and above is ignored, as the peak is very small. This suggests that the column starts to become overloaded with sodium benzoate between 100 and 200µl but significantly overloaded between 200 and 750µl.
The values for the symmetry of the peaks for the three compounds do not mirror those for efficiency. There does not seem to any trend.
Above 1000µl the values for efficiency increase dramatically and the peaks start splitting so are regarded as anomalous. Perhaps the reason is an overloaded column.
COLUMN 2: 100 × 4.6mm column of Genesis 300Å 4µ C18
Set volume – concentration varied
- The procedure is the same as in experiment 1.A and the same solutions were used
Flow rate: 1ml/min
Response: 0.5
Run-time: 6mins
Wavelength: 254nm
Range: depends on concentration used (See chromatograms)
Volume: 5µl
RESULTS
DISSCUSSION
The peak for Acetone is very small and hardly distinguishable due to the large number of peaks around its retention time, including a strong negative peak, which could affect its efficiency. The acetone could have evaporated due to its high volatility. The presence of acetone is to give t = 0 and hence to calculate k` values and so the peak can be ignored as it will not affect the other peaks and their efficiency. The retention time for this unretained peak is 1.32 min.
The % change in efficiency is calculated relative to the 1 in 10000 concentration, which is set at 100% efficiency. The efficiency values in plates per meter all have standard error reproducibility. This accounts for the deviations in efficiency values at lower concentration, which would be expected to be constant.
The efficiency of this column remains fairly steady between 1 in 10000 and 1 in 20 for the three compounds. From the results there seems to be a small but significant decrease in efficiency for the compounds between the 1in 20 and 1 in 10 solutions. There is again a small decrease between the 1in 10 and 1in 5 dilutions and then a large decrease in efficiency between the 1 in 5 and the standard solution. These results suggest that the column starts to become overloaded between 1 in 20 and 1 in 10 but is significantly overloaded between 1 in 5 and the standard solution where the is a 44%, 52%, 54% change in efficiency for theophylline, caffeine and sodium benzoate respectively.
The symmetry of the peaks for all the compounds generally tends to decrease as the concentration is increased from the most dilute to the most concentrated solution. This indicates that the efficiency decreases as the peaks are becoming more asymmetric and the column is overloading. It could be suggested that the results for symmetry mirror those of efficiency. There is a significant decrease in symmetry between the 1 in 5 and the standard solution, where there is a large decrease in the number of plates/m. Most of the symmetry values are below 1, indicating that b>a and a tailing effect observed.
Set Concentration – Volume varied
- Procedure as in experiment 1.A except the concentration was kept constant and the volume varied. The same HPLC conditions as 2.B were used.
- Concentration – 1 in 800 solution
2µl, 5µl, 10µl, 20µl, 50µl, 100µl, 200µl, 350µl, 500µl
As in 1.B each volume was tried on a larger loop to see if there was any effect (See results table)
RESULTS
All the results for theophylline are regarded as anomalous due to the peak having a pronounced shoulder and a strong negative peak just before it. Thus, the number of plates/m for theophylline is very low and the symmetry has negative values (see chromatograms). The reason for the presence of the shoulder is unknown. It was first thought to be a contaminant on the column but the column was flushed with water and acetonitrile to no avail. An impurity in the sample could be another cause. This did not occur in the other columns, however.
Discussion
Retention time for unretained acetone is 1.32 min.
Again different size loops were used for each volume to see whether loop size had any effect. The above results show a significant decrease in efficiency when a larger loop is used for a small volume. The most accurate values for efficiency in plates/m are to be converted into % and plotted.
The % efficiency is calculated relative to the 5µl volume, as the peaks on the 2µl chromatogram are very small. Even though 2µl and 5µl injected on the 5µl loop has a higher efficiency value than that of the 2µl and 5µl on the 20µl loop, the latter values are more in line with the other values and are taken as the efficiency results.
The % efficiency for caffeine remains fairly stable from 5µl to 200µl but there is then a significant decrease of ~43% from 200µl to 500µl. This suggests that this is the concentration region in which the column becomes overloaded.
For sodium benzoate, the % efficiency again remains relatively constant up until 200µl although there is a decrease of ~10% between 100µl and 200µl and then a further decrease of ~35% from 200µl to 500µl. It could be argued that the column starts overload with sodium benzoate at a lower volume than caffeine but shows a significant decrease in efficiency in the same concentration region.
Symmetry values for the peaks tend to follow the same trend as efficiency- staying constant then dropping off at high volumes. This indicates that the peaks are becoming more asymmetric as the column is overloading.
COLUMN 3: 100 × 4.6mm column of Kromasil 5µ C18
Set Volume – Concentration varied
- Procedure as in experiment 1.A
Flow rate: 1ml/min
Response: 0.5
Run-time: 8mins
Wavelength: 254nm
Range: depends on concentration (See chromatograms)
RESULTS
Discussion
Again the peak for acetone is indistinguishable. Retention time for this unretained peak is 1.22 min.
For theophylline, the % efficiency is calculated relative to the 1 in 10000 dilution, which is set at 100% as it is in line with the other dilutions. For caffeine, it is measured relative to the 1 in 1000 solution because there is a large decrease in efficiency for the 1 in 10000 dilution as the peaks are very small. This will therefore be classed as an anomalous result and not plotted. The % efficiency for sodium benzoate however, is measured relative to the 1 in 500 because the peaks in the 1 in 10000, 1000 and 800 are very small even though a high sensitivity was used. A noisy baseline is thus present. These efficiency values do not correlate with the other dilutions and so can be classed as anomalous and not plotted.
Between the 1 in 10000 and 1 in 10 solution for theophylline, the efficiency of the column remains fairly steady before decreasing by ~18% between 1 in 10 and 1 in 5 dilution. There is then a large decrease of ~38% between the 1 in 5 and standard solution.
A similar trend is observed in the results for caffeine where there is a steady % efficiency between 1 in 1000 and 1 in 10 but then a decrease of ~10% between the 1 in 10 and 1 in 5 dilutions. A further rapid decrease in efficiency of ~44% occurs between the 1 in 5 and standard solution.
For sodium benzoate the efficiency of this column again remains fairly constant between 1 in 500 and 1 in 10 dilutions (ignoring 1 in 800,1000, 10000 as they show large fluctuations in efficiency) before decreasing by ~7% between 1 in 10 and 1 in 5 dilutions. A large decrease in % efficiency is evident between 1 in 5 and the standard of ~48%.
It could be suggested that the column starts to become overloaded with all three compounds between the 1 in 10 and 1 in 5 dilutions by analysing these results. It could be argued that the starting point for overloading with caffeine and sodium benzoate is within the 1 in 20 and 1 in 10 dilution range. This is because there is a ~5% and ~4% decrease in efficiency respectively. However, it is clearly evident that the column becomes significantly overloaded with all three compounds between the 1 in 5 and standard solutions.
It could be suggested that that the results for symmetry follow the trend of % efficiency as they remain fairly steady at low concentrations before decreasing slightly between 1 in 10 and 1 in 5 and significantly between the 1 in 5 and standard solutions. Thus, indicating that the peaks are becoming more asymmetric as the efficiency decreases and the column overloads. Theophylline shows a reduction in symmetry from 1.41 to 1.24 between 1 in 20 and 1 in 10 suggesting that the column is overloading on symmetry faster than efficiency. Most values for symmetry are greater than 1 indicating that b>a and band tailing is occurring.
Set Concentration – Volume varied
- The same procedure as in Experiment 1.A except the concentration was kept constant and the volume varied. The same HPLC conditions as 3.A were used.
- Concentration used was the 1 in 800 solution
2µl, 5µl, 10µl, 20µl, 50µl, 100µl, 200µl, 350µl, 500µl, 1000µl, 2000µl
Due to the time limitation, these volumes were run on loops, which most closely matched their actual volumes, to give the most accurate efficiency values.
RESULTS
Discussion
The peak for acetone is indistinguishable. Retention time for this unretained peak is 1.22 min.
The % efficiency for theophylline and caffeine is calculated relative to the 5µl volume, as the number of plates/m for 2µl is considerably smaller. For sodium benzoate it is measured relative 2µl volume as the plates/m value tends to be in line with the other volumes used.
For theophylline, the % efficiency remains fairly constant between 2µl and 50µl. In the chromatogram for 100µl, the integrator did not calculate the peak for theophylline. This result was repeated but the same occurred. There is no explanation for this. The efficiency values between 200µl and 500µl are fairly constant but are at a significantly increased value compared to the lower volumes. There is then a very large increase between 500µl and 1000µl. This result, a long with the 2000µl value, is anomalous and is not in line with the other results and so can be ignored. These results suggest that with theophylline the column does not become overloaded. Above 2000µl, the peaks start to split and the efficiency values become unreliable. There is no apparent explanation for this – perhaps it is a sign of overloading, but this does not follow what occurs at overloading in the other experiments.
The % efficiency for caffeine follows the same sort of trends. The results for caffeine and theophylline will therefore not be plotted, as they will show a horizontal line.
With sodium benzoate however, the efficiency remains steady between 2µl and 100µl but then decreases ~10% by 200µl, then 17% by 350µl before decreasing a further ~20% by 500µl. Above this volume the peak is split into two and so the efficiency values are unreliable. This suggests that with sodium benzoate, the column starts to overload between 100 and 200µl and then becomes significantly overloaded at volumes greater than 200µl.
The symmetry values do not follow the same trend as efficiency but are widely scattered.
COLUMN 4: 100 × 10mm column of Genesis 120Å 4µ C18
Set volume – concentration varied
- Procedure as in experiment 1.A
Flow rate: 4ml/min
Response: 0.5
Run-time: 8mins
Wavelength: 254nm
Range: depends on concentration used (See chromatogram)
- Due to the wider column being used, the standard concentration solution already made did not overload the column sufficiently. A more concentrated solution in (standard 2) was made up with the following concentrations of each compound:
1. Acetone
Mass = 0.0623g
Number of moles = mass = 0.0623 = 0.001072 moles in 10 ml
M.w 58.1
0.001072 × 100 = 0.1072 mol dm-3
2. Theophylline
Mass = 0.1210g
Number of moles = mass = 0.1210 = 0.000671 moles in 10 ml
M.w 180.2
0.000671 × 100 = 0.0671 mol dm-3
3. Caffeine
Mass = 0.1850g
Number of moles = mass = 0.1850 = 0.000953 moles in 10 ml
M.w 194.2
0.000953 × 100 = 0.0952 mol dm-3
- Sodium Benzoate
Mass = 0.6753g
Number of moles = mass = 0.6753 = 0.00469 moles in 10 ml
M.w 144.1
0.00469 × 100 = 0.469 mol dm-3
Using the above amounts of compounds was difficult to dissolve in the mobile phase (12% acetonitrile). So, to help each compound dissolve a few drops of acetonitrile was added before HPLC grade water was added. The solutions were also heated using a water bath to aid dissolving.
It was not possible to increase the concentration of the compounds any further as they were difficult to dissolve in 10 ml and the compound that did dissolve started to come out of solution by the next day. However, the dilutions were made before the compounds came out of the solution otherwise the concentration calculated would not be the true concentration.
RESULTS
DISCUSSION
The peak for acetone is not clear. Retention time for this unretained peak is 2.12 min.
For theophylline and sodium benzoate the % efficiency is measured relative to the 1 in 100 dilution as the peaks in the in 1in 1000 dilution are very small and the value is not in line with the others. This was not the case for caffeine as the 1 in 1000 dilution was close to the other values.
For all three compounds the efficiency of the column remains steady from the most dilute solution to the 1 in 5 dilution but then decreases by 17%, 19%, 14% between 1 in 5 and standard 1 with theophylline, caffeine and sodium benzoate respectively. There is then a slight decrease for theophylline and caffeine of 12% and 4% between standard 1 and standard 2 but an increase in efficiency of 6% for sodium benzoate.
This suggests that the column starts to overload between 1 in 5 and the standard solution, unlike the other 4 columns where there is a very large decrease in % efficiency between the 1 in 5 and standard solution. From the standard 1 to standard 2 there is another slight decrease for theophylline and sodium benzoate but the efficiency seems to be remaining steady again. This change in efficiency could be due to some change within he HPLC instrument. It is likely that the column has not become overloaded with the concentrations used, but as stated, the concentration could not be increased any further.
The symmetry of the peaks does decrease from the most dilute solution to the most concentrated solution with theophylline and sodium benzoate. This effect is not as dramatic as for the other three columns. The symmetry for caffeine remains fairly constant indicating that the peaks are remaining symmetrical, showing no sign of overloading at these highest concentrations. Most of the symmetry values are greater than 1 implying that b>a and a tailing effect is seen.
Concentration constant – volume varied
- Procedure as in experiment 1.A except the concentration is kept constant and the volume varied. The same HPLC conditions as 3.A were used.
- Concentration used – 1 in 800 dilution
2µl, 5µl, 10µl, 20µl, 50µl, 100µl, 200µl, 500µl, 1000µl, 2000µl
As in Experiment 3.B the volumes were run on the loops nearest to their actual volumes.
RESULTS
Discussion
Retention time for unretained acetone is 2.12 min.
The % efficiency values are calculated relative to the 10µl volume as the peaks on the 5µl chromatogram are very small and a high sensitivity was used producing a noisy baseline.
For all three compounds the % efficiency remains fairly stable from 5µl to 2000µl apart from an anomalous result at 100µl which has a lower efficiency. This could be due to a dead volume or leakage associated with the loop. A 5ml loop was available, however the results acquired from this were anomalous, as they seemed to have a significantly greater efficiency even though the peaks were starting to split. This again could be due to a faulty loop and unfortunately there were no others available. It can be concluded however, that this column does not overload before 2000µl and so larger volumes would be required.
Again the symmetry does not show the same trend as efficiency.
CONCLUSION
The general trend in the results is that by increasing the concentration and the volume of the sample injected to a certain value, a decrease in efficiency of the column occurs as the column becomes overloaded.
From the results, it can be concluded quantitatively that the first column to overload at increasing concentration is the 100 × 4.6mm column of Genesis C18 300Å 4µ, with a surface area of 120m2/g and a pore volume of 1 cc/gm. Next to overload is the 100 × 4.6mm column of Kromasil C18 5µ, with a surface area of 340m2/g and a pore volume of 0.9 cc/gm. Then is the 100 × 4.6mm column of Genesis C18 120Å 4µ, with a surface area of 300m2/g and a pore volume of 1 cc/gm. This column overloads around the same region as the Kromasil column but there is a larger percentage decrease in efficiency between the 1 in 5 and standard solution for the Kromasil column. The column that does not become fully overloaded at the highest concentration is the 100 × 10mm column of Genesis C18 120Å 4µ and so is overloaded the least with the solutions used.
The concentration of the three compounds at a 20% decrease in efficiency for the four columns was found from the graphs to determine quantitatively which of the four columns decreases in efficiency the most and therefore overloads first at lowest concentration:
The above values are approximate values as a curve of best fit was drawn on the graphs and errors will be associated with this. All values in log10 mol dm-3. It is evident from these results that column 2 overloads at the lowest concentration, then column 3 which is close to column 1 and then column 4.
It could be suggested that by increasing the concentration of the sample, the active sites of the column particles making up the packing become saturated more quickly leading to overloading.
The 100 × 4.6mm column of Genesis C18 300Å 4µ has the lowest surface area and so there are less active sites and the column would become overloaded at a lower concentration than that of a column of higher surface area. The Kromasil column becomes overloaded around the same concentration as the 100 × 4.6mm column of Genesis C18 120Å 4µ but with the Kromasil column there is a greater % change in efficiency. One difference between these columns is the fact that the Kromasil column has 5µ particles whereas the Genesis one has 4µ particles. It could be argued that with a higher concentration smaller particles would degrade faster than larger particles as their smaller active sites become saturated more rapidly. This would suggest that the Genesis column would overload before the Kromasil. However, 1µ is not a very significant difference and other factors are apparent. The Kromasil column has a greater surface area (340m2/g) compared to the Genesis column (300m2/g) and this would suggest overloading at a higher concentration as there are more active sites available to adsorb the increased concentration of sample. Pore diameter values vary from manufacturer to manufacturer. Therefore, Genesis and Kromasil values would differ and so cannot be compared.
The equation for pore diameter is:
PD = k × Pore volume
Surface area
Where k is a constant and varies from brand to brand. The two different columns may contain different pore material. The area of one may be greater so there are more active sites available.
The 100 × 10mm column of Genesis C18 120Å 4µ does become fully overloaded in this experiment. (The concentration of the solutions could not be increased any further as they were starting to come out of solution). This is likely to be due to there being more material in the column so that it is able to retain more sample. There are more active sites available and so a higher concentration could be used before all active sites become saturated and the column overloads.
The volumes of the 3 compounds at which there is a 20% decrease in efficiency was found from the graphs to determine quantitatively which of the four columns decrease in efficiency the most and therefore overload first:
The above values are in µl and are again approximate. It is evident that column 2 overloads at the lowest volume, then 3, then 1 and finally 4, which does not become overloaded.
From the volume experiments, the order of the columns overloading are the same as for the concentration experiments. It could be argued that by increasing the volume of the sample injected onto the column, the number of plates (N) would decrease. For example, if 1ml of sample was injected onto a 10cm long column, the sample is only “seeing” 9cm of the column and exits more quickly from the column. The top layer of the column packing is saturated, then the next and so on.
Using a column with a lower surface area, like the 100 × 4.6mm column of Genesis C18 300Å 4µ, compared to a larger surface area, such as the 100 × 4.6mm column of Genesis C18 120Å 4µ, would mean that there is less material and therefore active sites available to adsorb the sample. The column would fill more rapidly, filling the active sites present and become overloaded at lower volumes. On the other hand, with a larger surface area there is more material and active sites to adsorb the sample and the column would overload at higher volumes due to the greater capacity.
Smaller particles are likely to become overloaded at lower volumes compared to larger particles due to the larger active sites to adsorb the sample. This would suggest that the 100 × 4.6mm column of Genesis C18 120Å 4µ would overload at a lower volume than the Kromasil column which has 5µ particles. The results do not show this but in fact they are the other way around. This could be due to the difference in manufacturers as explained above.
The 100 × 10mm column of Genesis C18 120Å 4µ is over twice as wide as the other columns. It would contain twice as much material and active sites to adsorb the sample. It has a larger capacity than the other columns and this is evident in the results, as it does not become overloaded at the volumes used.
Symmetry is measured via:
b
a
Values for symmetry less than 1 indicate that a>b. Values greater than 1 indicate a<b.
According to the following plot of Cm against Cs:
If a>b, the sample is more in the mobile phase than should be so there are less vacant positions on the stationary phase for it to occupy and overloading occurs. On the other hand, if a<b, tailing of the peaks occur. In ideal situations, symmetry = 1 for a peak perfectly symmetrical.
Experimental problems
- To gain a more accurate point of overload, it would have been useful to use concentrations and volumes in-between the values where there was a decrease in efficiency. This would have given a more accurate value for the onset of overloading of the four columns. Due to the time limitations and the number of loops/syringes available, this was not possible.
- It was ensured that each experiment – either concentration or volume on a particular column was carried out using the same buffer solution, as this would affected retention times. The buffer had to be changed every few days in order to prevent the growth of microorganisms which would affect the efficiency of the columns.
-
Some sample loops in the volume experiments gave anomalous results. This effect was apparent in the results for 5ml loop, which were ignored. This hindered the experiment as the greatest volume that could be injected was the 2000µl which did not overload column 4 (100 × 4.6mm column of Genesis C18 120Å 4µ). The cause of this could be the presence of a dead volume within the loop due to the ferrel either being too short or too long. It could have been a faulty loop also.
-
Shoulders appearing on some peaks, in particular, the peak for theophylline in the volume variation experiment for column 2 (100 × 4.6mm column of Genesis C18 300Å 4µ) were difficult to explain. Firstly, a contaminant on the column was suggested – but the column was washed with water and acetonitrile for long periods of time to no avail. An impurity in the sample was also ruled out as it did not occur on any of the other columns and a new batch of sample was made up. Perhaps there was a blockage on the column itself? E.g. the frit.
- During the volume variation on column 4, a loop snapped inside the rheodyne valve. This meant that the valve was changed and this changed the conditions of the experiment. The number of plates/m increased dramatically and so the results already acquired for this column were repeated under the new conditions in order for them to be compared. The increase in efficiency suggests that the new valve was more efficient.
- Other peaks present in the chromatogram are regarded as impurities.
- Increasing the concentration of the samples any further posed a problem as they started to come out of solution. It was ensured that the solutions were made before this occurred.
- The scope of this project is extremely large and if time was not a limitation it could be extended further. Varying the sample solvent to increase concentration levels, pH and % organic of the mobile phase could be investigated. These parameters could then be optimized to give maximum yield and purity. Manual fractions may be collected and analytical samples run.
References
- Principles of instrumental analysis – 5ed. Skoog, Holler, Nieman.
- Practical HPLC methodology and application – Brian. A. Bidlingmeyer. Wiley 1997.
- Introduction to modern liquid chromatography – L. R. Synder, J. J Kirkland. Wiley 1994.
- Analytical chemistry – Gary D. Christian.
- Pitfalls and Errors of HPLC in pictures – Veronika R. Meyer.
- Journal of Chromatography – 363 (1986) 1-30, J. H Knox et al.
- Journal of Chromatography – 249 (1982) 231-238, M Verzele et al.
ACKNOWLEDGEMENTS
I would like to thank Dr. R. S Ward (the project supervisor) and Stanley Szajda (laboratory technician) for their kind help and support throughout the duration of the project. I would also like to direct my gratitude towards Jones Chromatography, represented by Dr. Neil Herbert, who provided the project, supplied all columns and samples and gave appreciated guidance during the seven-week period.
INDEX
Pages
1. Column 1 (100 × 4.6mm column of Genesis 120Å 4µ)
A. Concentration varied – volume constant
-Method 8-10
-Results 11-12
-Discussion 12
-Graphs -
B. Volume varied – concentration constant
-Method 13
-Results 14-16
-Discussion 16-17
-Graphs -
-
Column 2 (100 × 4.6mm column of Genesis 300Å 4µ)
- Concentration varied – volume constant
-Method 18
-Results 19-20
-Discussion 20-21
-Graphs -
- Volume varied – concentration constant
-Method 21
-Results 22-23
-Discussion 23
-Graphs -
-
Column 3 (100 × 4.6mm column of Kromasil 5µ)
- Concentration varied – volume constant
-Method 24
-Results 25-26
-Discussion 26-27
-Graphs -
- Volume varied - concentration constant
-Method 27
-Results 28-29
-Discussion 29
-Graphs -
-
Column 4 (100 × 10mm column of Genesis 120Å 4µ)
- Concentration varied – volume constant
-Method 30-31
-Results 32
-Discussion 33
-Graphs -
- Volume varied – concentration constant
-Method 33
-Results 34
-Discussion 35
-Graphs -