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• Level: GCSE
• Subject: Maths
• Word count: 11129

# Liquid chromatography is a technique used to separate components of a mixture to isolate them for further use in synthesis (preparative chromatography) and for identification.

Extracts from this document...

Introduction

## INTRODUCTION

Liquid chromatography is a technique used to separate components of a mixture to isolate them for further use in synthesis (preparative chromatography) and for identification. The separation is achieved by forcing the mixture over an immobilised chemical system in a column by means of a liquid solvent stream. The individual solutes in the mixture partition differently between the moving and immobilised phases due to different chemical interactions, and travel at different rates down the column. By the time the mixture exits the column, the solutes are spatially separated and can be collected and analysed.

The are two modes of chromatography: normal-phase and reverse phase chromatography. In normal-phase chromatography, the retention is governed by the interaction of the polar parts of the stationary phase and the solute. For retention to occur in normal phase, the packing must be more polar than t

Middle

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

1. ### Set volume – concentration varied

• The procedure is the same as in experiment 1.A and the same solutions were used
• HPLC conditions:

Flow rate: 1ml/min

Response: 0.5

### Run-time: 6mins

Wavelength: 254nm

Range: depends on concentration used (See chromatograms)

## RESULTS

• Theophylline:
 Dilution Concentration (mol dm-3) Log10 concentration RetentionTime (min) Efficiency (plates/m) Efficiency      (%) Symmetry[10%]   height Standard 0.045 -1.35 2.02 14777 32.6 0.76 1 in 5 0.009 -2.05 2.00 35014 77.2 1.02 1 in 10 0.0045 -2.35 2.00 35917 79.2 1.09 1in 20 0.0023 -2.64 2.00 40595 89.5 1.14 1 in30 0.0015 -2.82 2.00 40115 88.5 1.04 1in 50 0.0009 -3.05 2.00 37930 83.6 1.03 1in 80 0.00056 -3.25 2.00 40372 89.0 1.14 1 in100 0.00045 -3.35 1.98 40351 89.0 1.14 1 in 500 0.00009 -4.05 2.00 45064 99.4 1.08 1 in 1000 0.000045 -4.35 2.00 44142 97.3 1.23 1 in 10000 0.0000045 -5.35 2.00 45345 100 0.99
• Caffeine:
 Dilution Concentration (mol dm-3) Log10 concentration Retention Time (min) Efficiency(plates/m) Efficiency     (%) Symmetry[10%]    height Standard 0.064 -1.19 2.82 15201 31.4 0.87 1 in 5 0.013 -1.89 2.82 40149 82.9 0.86 1 in 10 0.0064 -2.19 2.87 44160 91.2 0.96 1 in 20 0.0032 -2.49 2.80 47876 98.9 0.99 1 in30 0.0021 -2.67 2.80 47659 98.4 0.98 1 in 50 0.0013 -2.89 2.80 47186 97.4 1.01 1 in 80 0.00080 -3.09 2.80 49032 101.2 0.94 1 in 100 0.00064 -3.19 2.78 47139 97.3 0.90 1 in 500 0.00013 -3.89 2.82 48501 100.1 1.10 1 in 1000 0.000064 -4.19 2.83 45349 93.6 1.17 1 in 10000 0.0000064 -5.19 2.82 48431 100 1.10
• Sodium Benzoate
 Dilution Concentration (mol dm-3) Log10 concentration Retention Time(min) Efficiency(plates/m) Efficiency     (%) Symmetry[10%]   height Standard 0.326 -0.50 4.20 18997 36.0 0.36 1 in 5 0.0632 -1.20 4.10 42439 89.5 0.72 1 in 10 0.00316 -1.50 4.13 50378 95.4 0.85 1 in 20 0.0158 -1.80 4.05 48856 92.5 0.81 1 in 30 0.01053 -2.00 4.05 51149 96.9 0.88 1 in 50 0.00632 -2.20 4.07 50928 96.5 0.89 1 in 80 0.00395 -2.40 4.03 53995 102.3 0.90 1 in 100 0.00319 -2.50 4.00 55448 105.0 0.99 1 in 500 0.000632 -3.20 4.12 56693 107.4 1.19 1 in 1000 0.000316 -3.50 4.17 48906 92.6 1.22 1 in 10000 3.16×10-5 -4.50 4.13 52794 100 0.98
##### 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.

1. ### 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
• Volumes used:

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

• Theophylline

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.

• Caffeine
 Volume (µl) Loop size      (ul) Retention time (min) Efficiency    (plates/m) Efficiency   (%) Symmetry(10%) height 2 5 2.48 43970 - 0.90 20 2.48 31439 - 0.73 5 5 2.48 43443 - 1.03 20 2.48 36326 100 1.04 10 20 2.48 36751 101.2 0.96 100 2.53 24889 - 0.63 20 20 2.50 37405 103.0 0.83 100 2.53 26327 - 0.84 50 100 2.58 32679 90.0 0.72 200 2.62 32805 - 0.77 100 100 2.63 34112 93.9 0.72 200 2.68 28778 - 0.68 200 200 2.78 32764 90.2 0.72 350 500 3.17 9937 27.4 0.71 500 500 3.28 17065 47.0 0.75 2000 3.08 16611 - 0.53
• Sodium benzoate
 Volume (µl) Loop size     (µl) Retention time (min) Efficiency   (plates/m) Efficiency      (%) Symmetry(10%) height 2 5 3.75 54360 - 1.02 20 3.78 34825 86.7 0.75 5 5 3.77 52779 - 1.00 20 3.78 40157 100 0.77 10 20 3.82 39996 99.6 0.96 100 3.82 30378 - 0.59 20 20 3.80 40436 100.7 0.76 100 3.83 33071 - 0.59 50 100 3.85 33372 83.1 0.63 200 3.92 33714 - 0.72 100 100 3.90 30827 76.8 0.59 200 3.97 25910 - 0.65 200 200 4.03 26656 66.4 0.68 350 500 4.13 8034 20.0 0.81 500 500 4.13 12702 31.6 1.42 2000 3.87 12146 - 0.72

## 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

1. ### Set Volume – Concentration varied

• Procedure as in experiment 1.A
• HPLC Conditions:

Flow rate: 1ml/min

Response: 0.5

Run-time: 8mins

Wavelength: 254nm

Range: depends on concentration (See chromatograms)

RESULTS

• Theophylline:
 Dilution Concentration (mol dm-3) Log10 concentration Retention Time (min) Efficiency(plates/m) Efficiency    (%) Symmetry[10%]   height Standard 0.045 -1.35 2.13 21144 37.0 0.93 1 in 5 0.009 -2.05 2.13 42694 75.0 0.92 1 in 10 0.0045 -2.35 2.12 52724 92.6 1.24 1 in 20 0.0023 -2.64 2.18 52298 91.9 1.41 1 in 30 0.0015 -2.82 2.20 49248 86.5 1.03 1 in 50 0.0009 -3.05 2.20 51631 90.7 1.11 1 in 100 0.00045 -3.35 2.20 54224 95.2 1.31 1 in 500 0.00009 -4.05 2.20 57921 101.7 1.33 1 in 800 5.6×10-5 -4.25 2.18 52187 91.7 1.77 1 in 1000 4.5×10-5 -4.35 2.20 57194 100.4 1.36 1 in 10000 4.5×10-6 -5.35 2.20 56938 100 1.16
• Caffeine:

Conclusion

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

• Introduction                                                                                                1- 6
• Objectives                                                                                                   7
• Apparatus                                                                                                    7
• Procedure

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                                                                                                         -

1. Column 2 (100 × 4.6mm column of Genesis 300Å 4µ)
1. Concentration varied – volume constant

-Method                                                                                                        18

-Results                                                                                                         19-20

-Discussion                                                                                                   20-21

-Graphs                                                                                                          -

1. Volume varied – concentration constant

-Method                                                                                                        21

-Results                                                                                                         22-23

-Discussion                                                                                                   23

-Graphs                                                                                                           -

1. Column 3 (100 × 4.6mm column of Kromasil 5µ)
1. Concentration varied – volume constant

-Method                                                                                                         24

-Results                                                                                                          25-26

-Discussion                                                                                                    26-27

-Graphs                                                                                                              -

1. Volume varied - concentration constant

-Method                                                                                                           27

-Results                                                                                                            28-29

-Discussion                                                                                                      29

-Graphs                                                                                                               -

1. Column 4 (100 × 10mm column of Genesis 120Å 4µ)
1. Concentration varied – volume constant

-Method                                                                                                           30-31

-Results                                                                                                            32

-Discussion                                                                                                      33

-Graphs                                                                                                             -

1. Volume varied – concentration constant

-Method                                                                                                           33

-Results                                                                                                            34

-Discussion                                                                                                      35

-Graphs                                                                                                             -

• Conclusion                                                                                                       36-39
• Experimental problems                                                                                    39
• References                                                                                                        40

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