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 TITLE:

High Performance Liquid Chromatography (HPLC): Method

Development

OBJECTIVE:

To study development for optimizing a separation of a mixture of 5 compounds

which are caffeine, acetone, methyl benzoate, phenotate and phenanthrene using

HPLC by varying the mobile phase composition.

ABSTRACT

A further refinement to HPLC method has been developed and validated for the

determination of 5 compounds which are caffeine, acetone, methyl benzoate,

 phenotate and phenanthrene by varying the mobile phase composition during the

analysis; this is known as gradient elution. Gradient elution is where the mobile

 phase compostion is change with time during the separation. The method was

intended to decrease the retention of the later-eluting components so that they elute

faster, giving a narrower (& taller) peaks for most components and improves the

 peak shape for tailed peaks. In Optimum resolution was achieved by gradient

elution on an analytical column with the mobile phase consisting of a 

acetonitrile:water (20:80 v:v at a flow rate of 2.0 mL/min. The retention times of

caffeine, acetone, methyl benzoate, phenotate and phenanthrene were about 0.783,

0.864, 2.049, 2.434 and 3.717 min, respectively. Data acquisition was carried out

using a photo diode array detector in the wavelength 254 nm. Extraction of

chromatograms was carried out by timed wavelength. Data obtained in these

studies indicated that the method was suitable for the intended purpose. 

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PROCEDURE

A.INSTRUMENT SET UP

Detector wavelength: 254nm

Flow rate: 2.00 mL min-1 

Mobile phase: acetonitrile: water

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B. EFFECT OF MOBILE PHASE ONHPLC SPERATION

The instrument was set up with mobile phase ratio of acetonitrile:water (50:50 v:v)and the sample is injected into the column.

This ratio is repeated for three times to

verify the results.

After that, the mobile phase composition ischange to acetonitrile:water (70:30 v:v) and

the sample is injected into the column.

This ratio is also repeated for three times toverify the results.

Then, the resolution for both composition iscalculated and compared .

The suitable composition of mobile phaseof these copmounds is identified.

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D. SEPERATION USING GRADIENTELUTION

Based from the seperation above, thegradient elution seperation is performed

to improved the column efficiency.

The suitable ratio of mobile phase is setup and the sample is injected into the

column.

This method is repeated until the sitableratio of mobile phase is identified and all peak is seperated nicely and short resist

time.

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RESULT 

All the chromatogram for this experiment has been analyzed and it is attached

 behind the lab report. The resolution for isocratic elution for mobile phase

composition ACN:H2O (50:50 v:v) is tabulated in Table 1.1, 1.2, 1.3 and for

mobile phase composition ACN:H2O (70:30 v:v) is tabulated in Table 2.1, 2.2 and

2.3 . While, the average resolution of both mobile phase compositions is tabulated

in Table 3. Lastly, resolution for gradient elution is tabulated in Table 4. 

Resolution (Isocratic elution)

Mobile phase composition: ACN:H2O (50:50 v:v)

Peak Calculation Resolution, R s 

1-2

 

=2.912

2-3

 

= 22.618

3-4

 = 15.776

4-5

 

= 37.441

Table 1.1: Resolutions run 1

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Peak Calculation Resolution, R s 

1-2

 

= 2.161

2-3

  = 23.273

3-4

 

=17.011

4-5

 

= 39.873

Table 1.2: Resolutions run 2

Peak Calculation Resolution, R s 

1-2

 

= 2.286

2-3

 

= 25.493

3-4

 

=18.525

4-5

  = 42.599

Table 1.3: Resolutions run 3

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Mobile phase composition: ACN:H2O (70:30 v:v)

Peak Calculation Resolution, R s 

1-2

 

= 1.558

2-3

 

= 9.396

3-4

 

= 6.878

4-5

 

= 17.040

Table 2.1: Resolutions run 1

Peak Calculation Resolution, R s 

1-2

 

= 1.799

2-3

 

= 12.213

3-4

  = 9.492

4-5

 

= 28.463

Table 2.2: Resolutions run 2

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Peak Calculation Resolution, R s 

1-2

 

= 1.571

2-3

  = 10.050

3-4

 

= 7.425

4-5

 

= 17.656

Table 23: Resolutions run 3

Mobile phase composition Average Resolution, R s

ACN:H2O (50:50 v:v) 2.21

ACN:H2O (70:30 v:v) 1.64

Table 3: average resolution of both mobile phase compositions

Resolution (Gradient elution)

Peak Calculation Resolution, R s 

1-2

 

= 1.744

2-3

 

= 22.854

3-4

 

= 6.644

4-5

 

=18.744

Table 4: Resolutions for gradient elution

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DISCUSSIONS

HPLC is a technique for separation, identification and quantification of

components in a mixture. It is especially suitable for compounds which are not

easily volatilized, thermally unstable and have high molecular weights.

High performance liquid chromatography is basically a highly improved

form of column chromatography. Instead of a solvent being allowed to drip

through a column under gravity, it is forced through under high pressures of up to

400 atmospheres. That makes it much faster. It also allows us to use a very much

smaller particle size for the column packing material which gives a much greater

surface area for interactions between the stationary phase and the molecules

flowing past it. This allows a much better separation of the components of the

mixture. The other major improvement over column chromatography concerns the

detection methods which can be used. These methods are highly automated and

extremely sensitive.

According to………….  for the column and the solvent, there are two

variants in use in HPLC depending on the relative polarity of the solvent and the

stationary phase which is normal phase HPLC and reversed phase HPLC.

 Normal phase HPLC is essentially just the same as in thin layer

chromatography or column chromatography. Although it is described as normal, it

isn't the most commonly used form of HPLC. The column is filled with tiny silica

 particles, and the solvent is non-polar - hexane, for example. A typical column has

an internal diameter of 4.6 mm (and may be less than that), and a length of 150 to

250 mm. Polar compounds in the mixture being passed through the column will

stick longer to the polar silica than non-polar compounds will. The non-polar ones

will therefore pass more quickly through the column.

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 Next, for the reversed phase HPLC, in this case, the column size is the same,

 but the silica is modified to make it non-polar by attaching long hydrocarbon

chains to its surface typically with either 8 or 18 carbon atoms in them. A polar

solvent is used for example, a mixture of water and an alcohol such as methanol.

Hence, there will be a strong attraction between the polar solvent and polar

molecules in the mixture being passed through the column. There won't be as much

attraction between the hydrocarbon chains attached to the silica (the stationary

 phase) and the polar molecules in the solution. Polar molecules in the mixture will

therefore spend most of their time moving with the solvent.

 Non polar compounds in the mixture will tend to form attractions with the

hydrocarbon groups because of van der Waals dispersion forces. They will also be

less soluble in the solvent because of the need to break hydrogen bonds as they

squeeze in between the water or methanol molecules, for example. They therefore

spend less time in solution in the solvent and this will slow them down on their

way through the column. That means, now it is the polar molecules that will travel

through the column more quickly.

The liquid phase is pumped at a constant rate to the column packed with the

stationary phase. Before entering the column the analysis sample is injected into

the carrier stream. On reaching the column the sample components are selectively

retained on the basis of physic chemical interactions between the analyte molecules

and the stationary phase. The mobile phase moving at a steady rate elutes the

components based on the operating conditions. Detection techniques are employedfor detection and quantification of the eluted components.

The HPLC schematic diagrams are shown in Figure 1 and HPLC machine is

shown in Figure 2. In HPLC system, there are about eight important components.

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Some of the significance and role of each component part of the HPLC system is

discussed.

Figure 1: HPLC schematic diagram

Figure 2: HPLC machine

First of all, the mobile phase. Mobile phase serves to transport the sample to the

system. Essential criteria of mobile phase are inertness to the sample components.

Pure solvents or buffer combinations are commonly used. The mobile phase should

 be free of particulate impurities and degassed before use.

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 Next is mobile phase reservoir. These are inert containers for mobile phase

storage and transport. Generally transparent glass bottles are used so that so as to

facilitate visual inspection of mobile phase level inside the container. Stainless

steel particulate filters are provided inside for removal of particulate impurities in

the mobile phase if any.

Other than that, variations in flow rates of the mobile phase effect elution

time of sample components and result in errors. Pumps function in providing

constant flow of mobile phase to the column under constant pressure and to

 pressurize the liquid mobile phase

 Next is the injector. Injectors are used to provide constant volume injection

of sample into the mobile phase stream. Inertness and reproducibility of injection

are necessary to maintain high level of accuracy.

Column contains the bed of stationary phase. It is a vital component and

should be maintained properly as per supplier instructions for getting

reproducibility separation efficiency run after run.

Other than column, the column oven is also important part. The variation of

temperature during the analytical run can result in changes of retention time of the

separated eluting components. A column oven maintains constant column

temperature using air circulation. This ensures a constant flow rate of the mobile

 phase through the column

A detector gives specific response for the components separated by the

column and also provides the required sensitivity. It has to be independent of any

changes in mobile phase composition. It is function to detect the presence of

components as they exit the column and lastly the recorder. The recorders function

to record the detector signal.

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Modern HPLC systems are computer based and software controls

operational parameters such as mobile phase composition, temperature, flow rate,

injection volume and sequence and also acquisition and treatment of output.

Those are the main parts of a basic HPLC system more specialized

equipment might also have solvent selection valves, vacuum degasser, auto

samplers, column switchers, pre or post column derivatization and fraction

collectors.

As we mention earlier, there are two variants which is normal phase

chromatography and reversed phase chromatography. For this analysis, we used

reversed phase chromatography. In reversed phase chromatography, the stationary

 phase is non polar and the mobile phase is relative polar. The most polar

component will elute first, and increasing the mobile phase polarity increase the

elution time. Hence, because of the sample components interact with both the

stationary phase and the mobile phase the method development tends to be more

complex in liquid chromatography

Interactive mobile phase requires proper equalization intermolecular forces

among the three members in the separation process which is the solute, the mobile

 phase and the stationary phase in other to get successful chromatography. These

intermolecular forces are describes in term of the relative polarity of the reactants.

The polarities of various analytes functional groups are hydrocarbon

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 phase usually cannot compete successfully for the sample components; the

retention time becomes shorter for practical application. When the situation where

the polarity of the analyte and the stationary phase are too alike and totally

different from the mobile phase, thus the retention time becomes too long.

Due to theories of mobile phase and stationary phase interaction at any given

set of sample component are impacted, and at best, we can only narrower the

choice of the stationary has to a general type. Hence, because of this choice, we

then can perform a series of set trial and error experiment until a satisfactory

separation is identified.

During this experiment, a High Performance liquid chromatography (HPLC)

Agillent G1311A equipped with DA detector, 5 mm reverse phase C18 column and

10 microliter sample loop was used. At flow rate 2.0 ml/min and detector

wavelength at 254nm, the mobile phase ratio was set up at 50% acetonitrile and

50% water at the beginning at the experiment in other to analyze and observe the

effect of mobile phase composition on the chromatography separation. After the

standard mixture is injected, the process is run and the peak obtained is analyzed.

The process is repeated 3 times to get ideal result. Next, the resolution of the

 process is identified by calculating using formula below:

Where t is the retention time and W is the peak width.

After performing calculation for resolution as shown in Table 1.1, 1.2, and 1.3 we

get average resolution of 2.213.

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 Next, with the same requirement of flow rate and detector wavelength, the

mobile phase composition was changed to 70% acetonitrile and 30% water. The

same steps have been done and the process is also repeated for 3 times to get ideal

result. After performing calculation for resolution as shown in Table 2.1, 2.2, and

2.3 we get average resolution of 1.643.

Resolution describes the ability of a column to separate the peaks of interest,

and so the higher the resolution, the easier it is to achieve baseline separation

 between two peaks. Resolution takes into consideration efficiency, selectivity and

retention. It is useful to relate the resolution to the number of plates in the column,

the selectivity factor and the retention factors of the two solutes:

One can improve resolution by improving any one of these parameters. To

obtain high resolution, the three terms must be maximized. An increase in N, the

number of theoretical plates, by lengthening the column leads to an increase in

retention time and increased band broadening which may not be desirable. Instead,

to increase the number of plates, the height equivalent to a theoretical plate can be

reduced by reducing the size of the stationary phase particles.

It is often found that by controlling the capacity factor, k', separations can be

greatly improved. This can be achieved by changing the composition of the mobile

 phase (in Liquid Chromatography).

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The selectivity factor, α, can also be manipulated to improve separations.

When α is close to unity, optimising k' and increasing N is not sufficient to give

good separation in a reasonable time.

A value of 1 is the minimum for a measurable separation to occur and to

allow adequate quantitation. A value of 0.6 is required to discern a valley between

two equal-height peaks. Values of 1.7 or greater generally are desirable for rugged

methods. A value of 1.5 is considered to be a baseline separation and ensures the

most accurate quantitative result.

By comparing the average resolution for both mobile phase compositions,

we can see that with mobile phase ratio of 70% acetonitrile and 30% water gives

the best resolution which is 1.64. It is indicate that the resolution is good and

efficiency of separation is increase. Meanwhile, for the mobile phase ratio of 50%

acetonitrile and 50% water, the resolution is 2.21 shows that it has good resolution,

 but the efficiency of the separation is very weak.

After that, the components in standard mixture is identified when each of the

component is injected individually by using mobile phase compositions of 70%

acetonitrile and 30% water as the best composition baseline separation. The

compound in the standard mixture is identified by comparing the retention time of

standard mixture with the retention time of individual compound. From the result

obtained, peak 1 indicate compound of caffeine, peak 2 is acetone, peak 3 is

methyl benzoate, peak 4 is phenatole and the last peak is phenantrene.

Other than that, the rate theory of chromatography / Van Deemter equation

is related in HPLC broadening peak. Below are Van Deemeter equations:

H = A + B/v + Cv

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In HPLC the B term can be neglected due to diffusion is 100 times less in

liquids than in the gas. While the C term is the largest contribution to H. Consider

the following mobile phase mass transfer coefficient:

CMv = (f M(k’)d p2/DM)v

Where d p is particle diameter of packing and DM is mobile phase diffusion

coefficient. CMv is less if d p is smaller hence greater surface area or the solute

diffusion coefficient in the mobile phase, DM is larger.

 Next, a gradient elution separation method is performed in other to improve

te efficiency of the column. Meaning that, isocratic elution is performed with a

single solvent or constant solvent mixture. If one solvent does not provide

sufficiently rapid elution of all components, then gradient elution can be used.

In this case, by increasing amount of water was added to acetonotrile to

create a continuous gradient. In this experiment, using acetonitrile and water

gradient, the more hydrophobic components will elute when the mobile phase

consist mostly of acetonitrile which giving a relatively hydrophobic mobile phase.

The more hydrophilic compounds will elute under conditions of relatively low

acetontrile and high water. Often a series of test are performed on the analyte and

the number of the trial runs has been done in other to find the HPLC method which

gives the best separations peak. From the gradient elution method, the average

resolution obtained is 1.74 and it is indicate as good resolution and efficiency of

separation is high.

From the chromatogram obtain from both method which are isocratic elution

method and gradient elution method, there are some differences can be obtain from

the separation condition. In isocratic elution method, peak width increases with

retention time linearly according to the equation for N, the number of theoretical

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 plates. This leads to late eluting and peaks get a little bit flat and broad. In isocratic

elution method, the retention times of caffeine, acetone, methyl benzoate,

 phenotate and phenanthrene were about 0.739, 0.812, 1.318, 1.763 and 3.864 min.

Meanwhile, the gradient elution method decreases the retention of the later

eluting components so that they elute faster, giving narrower, taller and sharp

 peaks for most components. The retention times of caffeine, acetone, methyl

 benzoate, phenotate and phenanthrene were about 0.783, 0.864, 2.049, 2.434 and

3.717 min. This also improves the peak shape for tailed peaks, as the increasing

concentration of the organic eluent pushes the tailing part of a peak forward. This

also increases the peak height which where the peak look sharp which is important

in trace analysis.

Thus, the gradient elution method gave a shorter overall analysis with

similar resolution of the critical pair compared to isocratic elution without

sacrificing repeatability in retention time, peak area and peak height.

From the result, it shows that almost the entire peak is well separated from

the neighbor compound. But what was happened is only the peak 1 and the peak 2

is not well separated. Peak 1 indicates the caffeine compound and the peak 2

indicate acetone compound. The mobile phase composition used is 20%

acetonitrile and 80% water. It shows that, just a bit separation has occurred. As we

know, the quantitative analysis in separation method depends upon direct

relationship between the area under a peak or height in the chromatogram and the

amount of compound corresponding to that peak in the analyzed sample.

Therefore, each peak should be totally resolved from any neighboring peaks.

There are some factors leads to problem stated above. The mobile phase

must be degassed properly. It is because mobile phases entrap air from the

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atmosphere and this trapped air gets released as small bubbles under high pressures

encountered during the HPLC analysis. Such bubbles can lead to noise in detector

response or hinder flow of mobile phase through columns. In order to overcome

such problems degassing of mobile phase becomes essential. It is require a very

clean & pure HPLC grade solvent to prevent column degradation with impurities

& to minimize detector background signals from contaminants (usually UV

transparent). The gas that interference the HPLC analysis, such as N2, O2, and CO2.

The gas bubbles create difficulties with pumps destabilize pressure, columns bad

separation & detectors. Therefore we need to degasse the mobile phase in other to

get good analysis.

There are few methods in degassing the mobile phase. Vacuum devices

(vacuum applied to headspace above solvent). Ultra sonication (high frequency

vibration drives gasses out of solvent) Heating (decreases solubility of gases). He

sparging. Sparging is a process in which dissolved gases are swept out of a solvent.

There are some step is carefully taken when doing the experiment. When

injecting the sample into the loop, the sample volume is no more than volume

indicated on a loop. Besides, sample injection only with flat end needle to prevent

damage to the injection port.

Other than that, the syringe is washed at least 5 times with washing solution

and wash 3 times with sample before the sample is inejcted to the column to get

desire peak and no contiminants. When filling the syringe with the sample, the

maximum value should be 20μL. 

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CONCLUSION

From the experiment, the concept and method development of optimizing aseparation of a standard compounds using hplc by varying the mobile phase

composition is studied and determined.

REFERENCES

1.  Skoog, Hooler and Nierman, 5th Editon. Principles of Instrumental Analysis.

Thomson Learning 1998.

2. 

 Nor’ ashikin, Ruziyati and Mardiana, 2nd Edition. Analytical Separation

Methods Laboratory Guide