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HPLC

DETERMINATION OF CAFFEINE IN BEVERAGES BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

READING

Skoog D. A., Holler F. J., and Crouch S. R., Principles of Instrumental Analysis, 6 th edition, Harcourt Brace College Publishers, 2007. Chapters 26 and 28.

A. INTRODUCTION

Liquid chromatography (LC) refers to chromatography in which the mobile phase

is a liquid.

The four basic types of LC are partition chromatography; adsorption or

liquid-solid chromatography; ion exchange chromatography and size exclusion/ gel chromatography. They differ in the exact nature of the stationary phase, thus the mechanism/ processes insuring differential retention of analytes. Early LC utilized long glass columns with wide diameters (1 to 5 cm), and solid support particles of diameter in

the 150- to 250-µm range to insure reasonable flow rates (still less than 1mL per minute). Under these conditions, separations could take up to several hours. High-performance liquid chromatography (HPLC) was developed to increase speed and efficiency in liquid chromatography. Decreasing the size of the solid support material increased efficiency; i.e. decreases the height of theoretical plates. In the van Deemter equation [1], covered in the GC module,

H

=

A

+

B

+

Cu

=

A

+

B

+ (

C

 

+

C

 

)

 
 

S

M

u

 

u

u

the C coefficient which relates the linear velocity of the mobile phase to mass transfer

between phases, can be expressed as a sum of two coefficients

C

S

and

C

M

, related to

the stationary and mobile phase respectively. The

C

  • M coefficient is directly

proportional to the square of the diameter of the particles, leading to the conclusion that a

decrease in the size of the particles of the stationary phase supporting material will result in the decrease of the theoretical plate height ( H ). However, use of smaller size particles (3-10 µm) requires high pumping pressures (several thousands psi) for achieving separation within reasonable time periods. Whereas in gas chromatography the mobile phase does not interact with the analytes and serves only to transport analytes through the column, in liquid chromatography, the mobile phase interacts with the analyte, thus plays a very important role in affecting separation parameters such as retention times and resolution. The role of the mobile phase on separation adds versatility, flexibility and the range of forces that can be exploited to achieve separation of various complex mixtures by liquid chromatography.

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In bonded phase liquid chromatography, the stationary phase is composed of a

relatively low molecular weight solvent-like molecule covalently bound to a solid support

particle that is packed in the column. The solid support in modern high performance

columns typically consists of 3 to 10 µm-diameter porous silica gel particles (5 µm in this

experiment). In reverse phase bonded phase (RPBP) chromatography, the bonded phase

is non-polar and the mobile solvent phase is polar. And, in normal phase bonded phase

(NPBP) chromatography, the bonded phase is polar, and the mobile solvent phase is non-

polar. The column in this experiment is packed with an octadecyl (C 18 ) reverse-phase

bonded stationary phase.

In chromatography, a component injected onto the column will be distributed

between the mobile, M, and the stationary, S, phases according to its affinity for both

phases. Thus, in reverse phase HPLC, a polar molecule that interacts more strongly with

the M phase will elute quickly from the column. In contrast, a non-polar molecule would

interact more strongly with the S phase and so would elute more slowly from the column.

The retention time of a component on the column is related to the capacity factor,

'

k

i

, for

a column, which is in turn related to the distribution coefficient,

between the S and M phases [1].

K

  • i , of the analyte i

   

[ ]

i

s

K

i

=

[i ]

M

k

'

=

n

i

,

s

= K

W

s

i

n

i M

,

 
  • i V

M

 
 

where

 

W

s

= weight of S phase in column (g)

 

V

M

= volume of M phase in column (L)

[

i

] s

= concentration of i in s, mol/g

 

[

i

] M

= concentration of i in M, mol/L

 
 

'

 

Experimentally,

k

  • i is determined from the retention time for component i,

t

R i

,

,

and from the void time,

t

  • 0 . The void time is usually obtained from the time required for

either the solvent peak, or an unretained component of the mixture, to elute from the

column.

'

k

  • i =

t

R i

,

t

0

t

0

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Use of solvent mixtures

HPLC

Ideally, in a separation, the capacity factors (k’) for all components should lie

between 2 and 5 to effect good baseline resolution of the peaks in a limited period of

time. For a given stationary phase the k' of a particular component can be controlled by

changing the polarity of the mobile phase. Tables of solvent polarity or elution strength

have been prepared [2, 3]. However, the exact order of elution strength will depend on

the stationary phase and the components examined. Fine control of elution strength of

the mobile phase is obtained by using binary and ternary mixtures of solvents. In reverse

phase HPLC the most common solvent mixtures are H 2 O and methanol (CH 3 OH) or H 2 O

and acetonitrile (CH 3 CN).

Use of solvent gradient elution

Unfortunately, in complex mixtures k' can vary from zero to, more than twenty

for a single solvent strength. This leads to the "general elution problem" where no one

set of conditions is effective in removing all components from a column in a reasonable

time period, while still attaining resolution of each component. Since solvent polarity has

a strong effect on k' in reverse-phase chromatography, it is convenient to use a binary

solvent mixture (e.g. H 2 O and CH 3 OH) consisting of two solvents of differing polarity,

and change the percentage of each in the mixture during the elution of the sample. This

is known as gradient elution chromatography. In gradient elution, the solvent

composition changes over time as shown in Figure 1.

Conc. of A in A&B
Conc.
of A
in A&B

Time

Figure 1. Solvent gradient of solvent A with solvent B.

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Thus, the mobile phase polarity, or elution strength, varies during the chromatogram.

Note that various gradient shapes can be applied. Gradient elution will not be used this

semester, however an example of its use is discussed below.

Gradient elution is customarily used to separate components of a homologous

series. A typical experiment in Instrumental Analysis laboratory involves separation of a

homologous series of para-hydroxybenzoate esters. A mixtures containing the methyl-,

ethyl-, propyl-, butyl…nonyl hydroxybenzoates is separable within reasonable time by

applying gradient elution. Use of gradient elution allows good resolution of the lower

molecular weight components, while eluting the octyl and nonyl esters in a reasonable

period of time.

The solubilization of a solute in a solvent involves intermolecular interactions of

the solute (i) and solvent (j) with energy E ij [4, 5]. For an organic molecule consisting of

several different functional groups, e.g. CH 3 -(CH) n -Y, it is possible to break the overall

interaction energy up into the sum of individual interaction energies,

E

ij

=

E

CH

3

,

j

+

nE

CH

2

,

j

+

E

y

,

j

Martin first showed [6] that the log of the distribution coefficient,

K

i

, for a solute

between two phases j and l is approximately proportional to the sum of the differences of

the individual interaction energies as illustrated below for

CH

3

(

CH

2

)

n

Y

.

log

K

i

α

(

E

CH

3

,

j

E

CH

3

,

l

)

+

(

n E

CH

2

,

j

E

CH

2

,

l

)

+

(

E

Y

,

j

E

Y l

,

)

The Martin equation thus predicts that if

E

CH

2,

j

>

E

CH

2,

l

,

log K

i

(and

log k

'

i

)

will increase as the number, n, of a certain functional group in a homologous series

increases. In reverse phase chromatography, the

E

CH

2,

S

>

E

CH

2,

M

since the mobile

phase is polar and the octadecyl phase is not. Thus the hydroxybenzoate methyl ester will

be less retained than the hydroxy-benzoate nonyl ester.

B. GOALS OF EXPERIMENT

In this experiment, high performance reversed phase liquid chromatography is used

to:

1)

Separate the various substances contained in four common beverages from caffeine.

2)

Determine the concentration of caffeine in these beverages.

Separation will be conducted under ISOCRATIC conditions (i.e. elution at constant

solvent composition). This experiment illustrates the power of HPLC in analyzing

components in complex mixtures. Traditional method for the determination of caffeine is

via extraction followed by spectrophotometric quantitation. HPLC allows for rapid

separation and quantitation of caffeine from the many other substances found in these

beverages including tannic acid, caffeic acid and sucrose.

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C. INSTRUMENT AND OPERATION

HPLC

A Varian Model 5000 Liquid Chromatograph interfaced to a variable wavelength

UV/ VIS detector (Varian UV-50) and a strip chart recorder will be used. Reading of this

section may be facilitated by consulting the diagrams of important components appended

at the end of this hand out.

Column

The column is a commercially packed 5-µm octadecyl (C 18 ) bonded phase column

with a length of 25 cm.

Detector

The UV-50 Variable Wavelength detector is a double beam manual

spectrophotometer, equipped with a deuterium and a tungsten lamp, and a

monochromator, to provide wavelengths from 200nm to 720 nm. The optical path length

is 1 cm and the sample flow cell volume is 8-µL. The wavelength of measurement will

be 254 nm, the optimum wavelength for caffeine determination.

Solvents and Pump

Two solvent reservoirs, one containing HPLC grade methanol and the other

HPLC grade water, are already connected to the pump in the chromatograph. A two way

valve (Purge valve) permits routing of the solvent upstream the column (for purging)

when open (clockwise rotation), or to the column (counterclockwise rotation) when

closed.

Injector

The chromatograph is fitted with an automatic VALCO sample injector

comprised of a sampling valve, an external 10-µL sample loop and a fill-port fitting

assembly. The injector is controlled by the microprocessor. A programmed event 4

injects the content of the sample loop onto the column, and a programmed event 0 returns

the valve to the load position.

Operation: general starting procedures

  • 1. Turn power on the HPLC module, the UV/VIS detector, the recorder and the attenuator (unit above the recorder).

  • 2. Select the wavelength (254 nm) and the bandwidth (4 nm) on the UV/VIS detector.

  • 3. Use the CRT display and Keyboard to check and change the current conditions of the instruments and to build operating programs. After turning on the HPLC, the display should read, Power recovered, Programs saved. Press the DSPL key. The Flow rate (1.5-mL/ min.), solvent composition (%B 100), and EVNT (0) settings will be displayed. FLOW, %, PRGM, RSVR and EVNT are always used with the TIME key.

  • 4. Always use the instrument gradient program in order to gradually change to the desired composition.

  • 5. The instrument should be on for at least 30 minutes to equilibrate.

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D. EXPERIMENTAL PROCEDURES

HPLC

D.1 Mobile phase, Standard and Samples preparation

  • 1. Mobile phase/ solvent

1 L of a 25%: 75% Methanol: Water mixture (% volume).

The TA should have prepared the mobile phase/ solvent before the beginning of the

lab. HPLC solvents should always be of high purity (HPLC grade or freshly distilled)

in order to prevent alteration of the column chemistry by strongly adsorbing

impurities. The TA should have filtered the solvents to remove particulate larger than

0.45 µm, in order to preserve pump and column life.

  • 2. Standards Pipette 5-, 10-, 15- and 20-mL portions of the stock 10-ppm caffeine solution provided into 25-mL volumetric flasks, and dilute to volume with the solvent mixture. Pipette 1-mL of the 100 ppm solution of acetaminophen provided into 25-mL volumetric flask, and dilute to volume with the 10-ppm solution of caffeine. The stock solutions are already filtered so the standards do not need filtration.

  • 3. Instant Coffee Put ~150-mL of hot tap water into a 250-mL Erlenmeyer and set the flask on a hot plate to boil (heat the indicated amount of water for the other sample at the same time). Spoon out the amount of instant coffee appropriate for one cup, about one rounded teaspoon (1.5 to 2.5 g), onto a tarred weighing paper and record the mass of coffee to the nearest 10 mg. Do not use more than 2.5 g. Once the water has boiled, stir in the coffee and set aside to cool. When the coffee has cooled, transfer it quantitatively to a 200-mL volumetric flask and make up to the mark with water.

  • 4. Tea Bring to boil about 400-mL of hot tap water in a 500-mL flask. Meanwhile, weigh one tea bag and one emptied tea bag. Once the water has boiled, prepare the tea as you normally would, recording the time you steep the tea to the nearest 0.5-minute. Once brewed, remove the tea bag. When the tea has cooled, transfer quantitatively to a 500-mL volumetric flask and dilute to the mark with tap water.

  • 5. Dilution and Preparation for Injection Prepare a 20-fold dilution of the tea and the two degassed cola beverages, by pipetting 5-mL of each into 100-mL volumetric flasks, and diluting to volume with the solvent. Pipette 5-mL of the coffee solution into a 250-mL volumetric flask and dilute to volume with the solvent to obtain 1:50 dilutions. Before injection, the dilute solutions of tea and coffee must be filtered to remove particulate! Filter only ~10-mL of your samples (for cleaning the sample loop and for injections). The filtered sample can be kept in 4-dram vials. The vials should be very clean (absolutely no particles).

Rinse twice with a few drops of filtered samples.

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D.2 ANALYSIS OF STANDARDS AND SAMPLES

  • 1. Before beginning, check the solvent composition (must be 25 % CH 3 OH, indicated by %B = 25 on the CRT screen) and the flow rate (must be 1.5-mL/ min.). Open the purge-valve (clockwise rotation) and purge the system for five minutes. Remember to close it after flushing (counterclockwise rotation). Make sure the entry for EVNT is 0. Press start on the chromatograph to start pumping the solvent to the column for 5 to 10 minutes. If the composition of the solvent is different from 25 % (e.g. 100 %), set a gradient program to change the composition. Enter TIME = 20 min., % = 25, followed by ENTER. Check that at TIME = 0, the value for EVNT is 0, and the value for % is

100 %. Start the pump. When the solvent composition is 25 %, do not stop the

pump, just move to the next two steps.

  • 2. While pumping, use the COARSE ZERO Knob on the detector to move the pen to the zero position on the recorder, with the attenuator set at 8. This sets the zero absorbance reading of the detector using our mobile phase as the blank.

  • 3. Cleaning the sample loop before injection. Rinse the syringe with several aliquots of the solvent then fill it with the solvent. Make sure the sample injector is in the load setting (i.e. EVNT = 0). Insert the syringe needle all the way into the port. Do not push too hard on the syringe; just make sure it is bottomed. Flush about 250-µL of mobile phase through to clean the loading passages (do the same to prevent cross-contamination between runs). Refill the syringe, wipe clean, reinsert, and flush in another 250-µL, making sure not to inject any air bubbles. The flushing syringe is removed while flushing is being completed so as to fill the special teflon sleeve filling to top. This procedure prevents any bubbles from being pumped into the loop. Rinse the syringe with several aliquots of the sample to be injected. Before injection, be sure there are no air bubbles in the syringe. Point the needle upwards, tap the syringe with your fingers, and then push the plunger to expel the air bubbles.

  • 4. Flush and fill the sample loop with one of your standard solutions.

  • 5. To practice filling the sample loop and injecting use the following program TIME 0.0 EVNT 0 TIME 1.0 EVNT 4 TIME 1.2 EVNT 0

  • 6. In order to optimize reproducibility; use one continuous fill of the sample loop. Therefore, use the 1000-µL syringe so as to have sufficient sample in the syringe for refilling the sample loop between injections; and use the following program to make multiple injections at set intervals without stopping the pump.

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TIME 0.0

EVNT 0

ENTER

TIME 1.0

EVNT 4

ENTER

(1 st injection after 1 minute)

TIME 1.2

EVNT 0

ENTER

(close injector valve)

TIME 2.0

EVNT4

ENTER

(2 nd injection, 1 minute after first injection)

TIME 2.2

EVNT0

ENTER

(close injector valve)

TIME 3.0

EVNT4

ENTER

(3 rd injection, 1 minute after 2 nd injection)

TIME 3.2

EVNT0

ENTER

(close injector valve)

TIME 4.0 EVNT4

ENTER

(4 th injection, 1 minute after 3 rd injection)

TIME 4.2 EVNT0

ENTER

(close injector valve)

  • 7. Press start to run the program DO NOT FORGET TO REFILL THE SAMPLE LOOP BETWEEN INJECTIONS (you have 48 seconds to do this). Check that the retention times are reproducible (±0.05 min) to ensure that the column is equilibrated. Then, use the same procedure to make four replicate injections of all the standards. between runs.

Remember to reset the program

  • 8. Use a single injection program to obtain one 'clean' chromatogram of the Coca-Cola sample.

  • 9. Next, make four replicate injections of the Coca-Cola sample using two minutes intervals between injections in the program.

    • 10. Repeat steps 8 and 9 to obtain one 'clean' chromatogram and four replicate runs for the other beverages.

    • 11. When you have finished your injections, program the instrument to gradually change the mobile phase composition to 100 % CH 3 OH

TIME = 0

% = 25

TIME = 20

% = 100

To erase previous values, use the same commands, then press DEL instead of

ENTER. Make sure you have erased all previous entries or switch to a new program.

  • 12. Flush the sample loop with the solvent.

  • 13. Turn off the detector, the recorder, the attenuator and the chromatograph.

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E. QUESTIONS

HPLC

  • 1. Plot the peak areas and peak heights calibration curves for caffeine using units of ppm for concentration. Use the triangulation method to obtain peak areas (peak height times peak width at one-half peak height).

  • 2. Use linear regression analysis to obtain the best straight line through the experimental points [1,7].

  • 3. Calculate the standard deviation on the slopes and intercepts as described in Appendix 1 of your text Book [1] or reference [7].

  • 4. From the calibration curves report the concentration of caffeine in the injected beverage samples, and in the original samples in ppm. Evaluate the overall standard deviation for each of your results.

  • 5. Report the % by weight of caffeine in each of the original solid materials.

  • 6. Calculate the capacity factor of caffeine using the retention time of acetaminophen as

t

  • 0 .

  • 7. Explain the rationale for using a reverse-phase C18 column for the determination of caffeine.

  • 8. Derive the relationship between retention time and capacity factor from the following relations:

v = L / t

  • 0 = average linear velocity of mobile phase

v

i

=

L

/

t

R i

,

=

φ

i M

,

×

v

= average linear velocity of i in mobile phase

φ

i

,

M

=

n

i M

,

n

i s

,

+

n

i M

,

F. REFERENCES

  • 1. Skoog D. A., Holler F. J., and Crouch S. R., Principles of Instrumental Analysis, 6 th Ed., Harcourt Brace & Company, Orlando, Florida, 2007, Chap. 26 and Chap.28

  • 2. Snyder L. R., Kirkland J. J., Introduction to Modern Liquid Chromatography, John Wiley, Toronto, 1979, Pp. 218-225.

  • 3. Karger B. L., Snyder L. R., Horvath C., An Introduction to Separation Science, John Wiley, Toronto, 1973, Pp. 271-274.

  • 4. Ibid. Pp. 55-57, 275-276.

  • 5. Giddings J. C., Unified Separations Science, John Wiley & Sons Inc., New York, 1991, Pp. 24-30.

  • 6. A.J.P. Martin A. J. P., Biochem. Soc. Symp. 3, 4 (1949).

  • 7. Skoog D. A., West D. M., Holler, Analytical Chemistry: An Introduction, 5 th Ed., Saunders College, Philadelphia, 1990, Chapter 4, Sec. 4B-3 to 22-6.

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