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EXERCISE NO. 6 HPLC ANALYSIS OF BENZOIC ACID AND CAFFEINE IN SOFTDRINK SAMPLE

REHANA V. LEBBE CHEM 137.1 1L

DATE SUBMITTED: NOVEMBER 16, 2016

ABSTRACT

In the experiment, the chromatograms of caffeine and mixed standards (caffeine and benzoic acid) were determined using UPLC. Optimization of parameters including flow rate, sample load and solvent system were done using chromatograms of mixed standards and caffeine. The parts of HPLC were also explained throughout this paper. Information such as retention factor, resolution and selectivity factor were calculated and their importance in column efficiency were explained. Also, HETP and N values were determined using the three known methods: tangent method, 5σ method and half-height method.

I.

INTRODUCTION:

High performance liquid chromatography (HPLC) is a chromatographic technique used to separate mixture of compounds. This method is usually used in biochemistry and analytical chemistry to identify, quantify and purify individual components of mixture. Generally, in liquid chromatography, sample mixture is eluted through the packed column with a mobile phase supported by high pressure from the pump. The components are separated from one another based on the interaction of these components with the stationary phase and the mobile phase. These separated components are detected at the exit of the column by a detector that can measure the amount of the components present. An output from this detector is known as a “chromatogram”. (Harvey, 2000)

ABSTRACT In the experiment, the chromatograms of caffeine and mixed standards (caffeine and benzoic acid) were

Figure 6.1 Typical HPLC unit.

In HPLC, narrow columns with internal diameters 2-80 mm are used. These columns are packed with particles having an average diameter of less than 50 microns (50 x 10 -6 m). Such columns require high pressures (1000-6000 psi) to maintain a convenient flow of the eluting solvent, usually in the range 0.1-10 mL/min, but occasionally higher. Resolution is considerably superior to that achieved with an ordinary column, in part because of the tight packing of the stationary phase, which reduces lateral diffusion and because of the large surface area of the packing. In comparison to HPLC, classical column

chromatography employs columns that are gravity operated which can take hours or days before all components are eluted from the column. Hence, HPLC has a better analysis time as well as resolution of chromatogram. (Harvey, 2000) HPLC is especially suited to the analysis of compounds not readily analyzed by gas chromatography. For example, thermally labile compounds can be analysed by HPLC at ambient temperatures as well as highly polar or nonvolatile compounds can be analysed. Sample treatment is often minimal since aqueous solutions can be used in HPLC. Since its inception in the late 1960's, HPLC has made significant practical impact on the areas of pharmaceutical, clinical, forensic, environmental and industrial research and development analysis. Preparative HPLC has also found an important use in the isolation and purification of various compounds. (Harvey, 2000)

For most users of HPLC, analyzing the performance of a chromatographic column consists of measuring the following three key performance parameters: the column efficiency (N or HETP), the retention factor (k), and the selectivity factor (α) for important pairs of analytes. Also, other parameters of column such as lifetime, chemical inertness, and sample load are also considered.

The objectives of the exercise were to optimize the following parameters: solvent system, best working flow rate and establish the appropriate sample load. The selectivity factor, capacity factor and column efficiency were calculated using the mixed standards.

II.

MATERIALS AND METHOD

A set of conditions for the Acquity Waters UPLC includes a C18 column with 17 µm pore size, 2.1x50 mm size. The temperature was set at 45at 15000 psi with wavelength detector at 210-400 nm.

The first run includes the injection of 10 µL of mixed standard at a flow rate of 0.6 mL/min with two different solvent system: 9:1 water: acetonitrile and 8:2 water: acetonitrile. Second run involves the injection of 10 µL of mixed standard using a solvent system 9:1 water: acetonitrile at two different flow rates: 0.3 mL/min and 0.60 mL/min. The third run involves the injection of 5 µL and 10 µL of mixed standard with a flow rate of 0.6 mL/min using a solvent system 9:1 water: acetonitrile.

III.

RESULTS AND DISCUSSION

Liquid chromatography basically employs the idea of the partitioning of the analyte between the sample-free phase known as mobile phase and another sample-free phase that remains fixed known as the stationary phase. Once components are eluted along with the mobile phase, interaction of the components with the stationary phase will determine its retention time on the column. This can be detected by a detector to produce a chromatogram. (Harvey, 2000)

Using the UPLC, mixed standards of benzoic acid and caffeine, caffeine and unknown sample was analyzed for their components. The first run involved the injection of 10 µL of mixed standard at a flow rate of 0.6 mL/min with two different mobile phase system: 9:1 water: acetonitrile and 8:2 water: acetonitrile. In this run, effect of changing the composition of mobile phase was observed.

Table 6.1. Data on the elution of caffeine using 9:1 water:acetonitrile solvent system.

Name

Retention Time

Area

%Area

Caffeine

0.637

234663

79.07

Table 6.2. Data on the elution of caffeine using 8:2 water:acetonitrile solvent system.

Name

Retention Time

Area

Height

Caffeine

0.482

2924870

2592665

As seen from the data gathered, four peaks were observed upon use of 9:1 water:acetonitrile in comparison to the one peak observed for the 8:2 water:acetonitrile. Also, caffeine was retained far longer with use of more polar mobile phase in comparison to the less polar mobile phase. This shows that the best solvent system was 9:1 water:acetonitrile because better separation of peaks can be observed for this solvent system.

Elution order of the analyte in HPLC is governed by polarity. In a normal-phase separation the least polar solute spends proportionally less time in the polar stationary phase and is the first solute to elute from the column. Retention times are controlled by selecting the mobile phase, with a less polar mobile phase leading to longer retention times. In reverse-phase chromatography, on the oher hand the stationary phase is nonpolar and the mobile phase is polar. The most common stationary phases used are organochlorosilane for which R group is an n-octyl (C8) or n-octyldecyl (C18) hydrocarbon chain. The mobile phase of RPLC are usually using a buffered aqueous solution. The pH of the mobile phase must be less than 7.5 since the silica substrate is subject to hydrolysis in basic solution. (Harvey, 2000)

To effect a better separation between two solutes it is often necessary to improve the selectivity factor, a. One approach is the adjustment of pH of mobile phase for ionic analytes. When a solute is a weak acid or a weak base, adjusting the pH of the aqueous mobile phase can lead to significant changes in the solute’s retention time. At more acidic pH levels, both weak acids are present as neutral molecules. They partition then favorably into the stationary phase resulting to fairly long retention times for the solutes. When the pH is more basic, the solutes present as their conjugate weak base anions are less soluble in the stationary phase and elute more quickly. (Harvey, 2000)

In the chromatography done in the experiment, the stationary phase is covalently bonded to silicate particles. Bonded stationary phases are attached by reacting the silica particles with an organochlorosilane of the general form Si(CH3)2RCl, where R is C18. The properties of a stationary phase are determined by the nature of the organosilane’s alkyl group. Since the R group of the stationary phase is nonpolar, the mobile phase used is highly polar. (Harvey, 2000)

Table 6.3. Data obtained for the mixed standards at 0.30 mL/min flow rate.

Name

Retention Time

Area

%Area

%Height

Benzoic Acid

0.515

94652

24.04

38398

Caffeine

1.192

228173

57.96

121632

Table 6.4. Data obtained for the mixed standards at 0.60 mL/min flow rate.

Name

Retention Time

Area

%Area

%Height

Benzoic Acid

0.278

41383

23.84

45978

Caffeine

0.64

113700

65.49

104783

The effect of different flow rates shows that longer flow rate results to better resolution of peaks in comparison to shorter flow rates with less resolution of peaks. Longer flow rates allow the components of the analyte to interact with the mobile and stationary phases longer. Once these components are retained, separation of the benzoic acid and caffeine is distinctive from the chromatogram. This is shown in the data gathered in the experiment. Comparing the data obtained in Table 6.3 and Table 6.4., it shows that components were retained longer in 0.30 mL/min flow rate than in 0.60 mL/min flow rate.

Table 6.5. Data obtained for the mixed standards with 10 µL sample load.

Name

Retention Time

Area

%Area

%Height

Benzoic Acid

0.278

41383

23.84

45978

Caffeine

0.640

113700

65.49

104783

Table 6.6. Data obtained for the mixed standards with 5 µL sample load.

 

Name

Retention Time

Area

%Area

%Height

Benzoic Acid

0.267

22370

25.8

27290

Caffeine

0.626

56375

65.01

52795

On the other hand, effect of the amount of sample load shows that the sample is introduced using a loop injector. Sampling loops are interchangeable, and available with volumes ranging from 0.5 mL to 2 mL. A syringe with a capacity several times that of the sampling loop is used to place the sample in the loop. Any extra sample beyond that needed to fill the sample loop exits through the waste line. After loading the sample, the injector is turned to the inject position. In this position the mobile phase is directed through the sampling loop, and the sample is swept onto the column. (Harvey, 2000) The effect of amount of sample load should not affect the peaks or resolution in the chromatogram. Results showed almost the same values of retention time of the 5 and 10 µL sample load.

Theoretically, polar stationary phase should retained the more polar component. Comparing the structure and polarity of benzoic acid and caffeine, it is shown that the benzoic acid is more polar compared to caffeine. Using the reverse-phase LC, the less polar compound is likely to be retained longer than the more polar compound. Thus, benzoic acid is expected to elute first than caffeine.

Table 6.4. Data obtained for the mixed standards at 0.60 mL/min flow rate. Name Retention Time
Table 6.4. Data obtained for the mixed standards at 0.60 mL/min flow rate. Name Retention Time

Figure 6.2. Structure of benzoic acid and caffeine

If there is no standard chromatogram present, other detectors could be used

such as mass

spectroscopy, diffractional refractometer or UV-vis spectroscopy. These detectors can result to other properties of the components of the analyte that can lead to the determination of their identity.

As the chromatographic run was observed, the peak widths of each component of the analyte continually increases. This is called as band broadening. Column efficiency is used to measure the extent of band broadening. (Harvey, 2000)

N= H L

As you see from the equation above, column efficiency increases upon the increase in the length of the column and decrease in the height of theoretical plate. The column efficiency calculated for the three different methods are the following:

  • a. Tangent method

 

N=16 (

  • b. 5σ method

t

R

W )

t R

W 4.4

¿

¿

N=25 ¿

  • c. Half-height method

t R

W 1/ 2

¿

¿

N=5.54 ¿

Table 6.7. Data calculated for N and HETP using three different methods.

Parameters

N

HETP

Tangent Method

1154.187

0.004015

5σ method

899.1841

0.005561

Half-height method

1245.37

0.004332

The main of goal of chromatography is to separate the components into chromatographic peaks. Resolution is then used to measure the degree of separation of two peaks in a chromatogram. (Harvey,

2000)

R s

= 2[t R, A

'

' t R, B

]

w A +w B

Using the mixed standards with the optimized parameters of 0.60 mL/min and sample load of 10 µL, the resolution calculated for the two peaks of benzoic acid and caffeine is 12.0666. A solute’s capacity factor (k) can also be determined from a chromatogram by measuring the column’s void time, t M , and the solute’s retention time, t R . k= t R t M

t M

The value of capacity factor indicates the solute’s distribution into the mobile phase and stationary phase. The column’s void time was 0.2. Using the mixed standard chromatogram with optimized parameters, the capacity factor of caffeine and benzoic acid are 0.39 and 2.2, respectively. The relative selectivity of a chromatographic column for a pair of solutes, on the other hand, is given by the selectivity factor, a, which is defined as:

a=

'

k A

'

k B

=

t

R, A

t M

t

R ,B

t M

The identities of the solutes are defined such that solute B always has the smaller retention time. Accordingly, the selectivity factor is equal to 1 when the solutes elute with identical retention times, and is

greater than 1 when t

R, A

is greater than

t

R, B

. (Harvey, 2000) The calculated selectivity factor for the

same chromatogram of mixed standard is 5.641026. These parameters are important in indicating the column efficiency of the instrument in separating the components of an analyte.

For the quantitative analysis of the unknown, single-point calibration was used to determine the amount of caffeine in diluted Mountain Dew.

Table 6.8. Data on the analysis of unknown in UPLC.

Name

Retention Time

Area

%Area

%Height

Benzoic acid

0.279

40780

31.3

46649

Caffeine

0.641

70217

53.78

64297

The calculations are shown in the Sample Calculations part of the paper and results showed that the amount of caffeine present in Mountain Dew is 4.10597 mg/L.

IV. SUMMARY AND CONCLUSION

The experiment involves the application of high-performance liquid chromatography specifically UPLC to mixed standards of caffeine and benzoic acid as well as caffeine alone. The instrument was first optimized for the following parameters: best solvent system, flow rate and sample load. Results showed that the following are the optimized parameters: 9:1 water:acetonitrile, 0.60 mL/min and 10 µL sample load.

Using the chromatogram of mixed standard with these optimum parameters, the capacity factor, selectivity factor and column resolution were calculated. These parameters are very important in determining whether the separation of the components of mixed standard was good and efficient. Results

showed that there was good separation of benzoic acid and caffeine. Also, elution order of the two were determined based on the polarity of these components. Since benzoic acid is more polar with low molecular weight, caffeine was eluted first and detected in the chromatogram followed by benzoic acid.

The chromatogram of caffeine was also used to calculate for the column efficiency, N, using the three known methods: tangent, and half-height method. The value obtained from these three methods were used to calculate for HETP. The following are the N and HETP obtained: 1154.1841, 899.1841, 1245.37 for N and 0.004015, 0.005561, 0.004332 for HETP.

The use of HPLC/UPLC is very essential since it can be used for analysis of many substances. Substances that cannot be analysed using gas chromatography can be analysed using HPLC. This is very useful in isolation and determination of identities of components of analyte.

  • V. SAMPLE CALCULATIONS

Calculation of void volume:

Void Volume= 0.17d 2 Lπ

4

Void Volume= 0.172.1 2 50π

4

=0.1177626006mL

Calculation of dead time:

Dead time= void volume

flow rate

Dead time= 0.1177626006mL

0.6mL/min

=0.196271001min

Using caffeine with 0.60 mL/min flow rate:

Calculate number of theoretical plates, N:

  • a. Tangent method

N=16(

0.637

cm )

0.3

2

N=1154.187

N=16 (

t

R

W )

  • b. 5σ method

t R

W 4.4

¿

¿

N=25 ¿

0.637

0.05 cm

¿

¿

N=5.54 ¿

  • c. Half-height method

t R

W 1/ 2

¿

¿

N=5.54 ¿

0.637

0.1cm

¿

¿

N=5.54 ¿

N=1245.37

Calculation for the HETP of 5 cm length column:

H= N L

H= 5 cm

1245.37

=0.004015

Using mixed standard with 0.60 mL/min flow rate and 10 µL sample injection:

Calculation for selectivity factor:

 

'

R, A

 

k A

=

t

t M

a=

'

k B

t

R ,B

t M

0.6400.2

 

a=

0.2780.2

=5.641026

Calculation for capacity factor:

 

Caffeine:

k= t R t M

 
 

t M

k= 0.2780.2

=0.39

 

0.2

Benzoic Acid:

k= 0.6400.2

0.2

=2.2

Calculation for resolution:

R

 

'

= 2[t R, A

' t R, B

]

s

w A +w B

 

R

= 2[2.20.39]

=12.0666

 

s

0.1+0.2

 

Quantitative analysis of caffeine in diluted Mountain Dew:

S=kC

Using the data of caffeine in Table 6.5. (mixed standard):

k= S

C

k=

  • 65.49 L =13.098 L/mg

5mg/

Using data of unknown in Table 6.8:

C= S k

C=

53.78

  • 13.098 L/mg =4.15097mg/L

VI.

REFERENCES

Harvey, D. (2000). Modern Analytical Chemistry. McGraw Hill.

Skoog, D. A., West, D. M., Holler, J. F., & Crouch, S. R. (2014). Fundamentals of Analytical Chemistry. Belmont, USA.