ANALYTICAL SEPARATION METHOD

CHM 510

EXPERIMENT 2:

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC): METHOD DEVELOPMENT

NAME : NUR HAZIMAH BINTI MAHAMAD POZI (2012448048)

PARTNER: SITI NORMALIA BINTI SULAIMAN (2012237674)

LECTURER:

GROUP: ASB2Ac

DATE OF EXPERIMENT: 9/10/2013

DATE OF SUBMISSION: 23/10/2013

TITLE

High Performance Liquid Chromatography (HPLC) Method Development

OBJECTIVE

To study development for optimizing a separation of a mixture of three compounds which are

standard mixtures of caffeine, phenatole and methyl benzoate using HPLC by varying the mobile

phase composition.

ABSTRACT

A further refinement to HPLC has been to vary the mobile phase composition during the

analysis; this is known as gradient elution. The gradient separates the analyte mixtures as a

function of the affinity of the analyte for the current mobile phase composition relative to the

stationary phase. This partitioning process is similar to that which occurs during a liquid-liquid

extraction but in continuous, not step wise. In this experiment, using water / acetonitrile 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 acetonitrile and high water. The choice

of solvent, addictives and gradient depend on the nature of the stationary phase and the analyte.

Often a series of tests are performed on the analyte and the number of trial runs may be

processed in order to find the HPLC method which gives the best separation of peaks.

INTRODUCTION

High performance liquid chromatography is the most widely used of all of the analytical

separation technique. It’s suitable for separating nonvolatile species or thermally fragile ones.

Partition chromatography is the most widely used of all the four types of liquid chromatography

procedure. It divides into two; normal-phase chromatography and reverse-phase

chromatography.

For this analysis we used reversed phase chromatography. In reverse-phase chromatography, the

stationary phase is non polar and the mobile phase is relatively polar. The most polar component

will elute first, and increasing the mobile phase polarity increase the elution time. Method

development tends to be more complex in liquid chromatography because the sample

components interact with both the stationary phase and the mobile phase. Successful

chromatography with interactive mobile phase requires a proper balance of intermolecular forces

among the three active participants in the separation process- the solute, the mobile phase, and

the stationary phase. These intermolecular forces are described qualitatively in term of the

relative polarity of three reactants. The polarities of various analytes functional groups in

increasing order are: hydrocarbon <ether <ester < ketones < aldehyde < amides < amines <

alcohols. Water is more polar compounds than compounds containing any of the preceding

functional groups.

Often in choosing a column for a partition chromatographic separation, the polarity of the

stationary phase is matched roughly with that of the analytes; a mobile phase of considerably

different polarity is then used for elution. This procedure is generally more successful than one in

which the polarities of the solute and mobile phase are matched but different from that of the

stationary phase. Here, the stationary phase often cannot compete successfully for the sample

components; retention time becomes too short for practical application. At the other extreme, of

course, is the situation where the polarity of the solute and stationary phase are too much alike

and totally different from that of the mobile phase. Here, the retention times becomes

inordinately long.

In summary, polarities for solute, mobile phase and stationary phase must be carefully blended if

good partition chromatography separation are to be realized in a reasonable time. Unfortunately,

theories of mobile phase and stationary phase interaction with any given set of sample

component are impacted, and at best, we can only narrow the choice of stationary phase to a

general type. Having made this choice, we then perform a series of set trial and error experiment

in which chromatogram are obtained with various mobile phase until a satisfactory separation is

realized. If resolution of the entire component of a mixture proves to be impossible, different

types of column may have to be chosen.

INSTRUMENTS

Liquid Chromatography (Agilent G1314A HPLC) equipped with UV detector, 5mm RP C18

column and 10 uL sample loop.

REAGENTS AND SOLVENTS

HPLC grade acetonitrile, deionized water, standard mixture of caffeine, phenatole, and methyl

benzoate (100 ppm).

SAMPLE

Standard mixture of caffeine, phenatole, and methyl benzoate (100 ppm)

ANALYTICAL PROCEDURE

1. Instrument set up

Detector wavelength : 254 nm

Flow rate : 1.5ml/min

Mobile phase : acetonitrile: water (50: 50 v/v)

2. Effect of mobile phase on Liquid Chromatography separation

a. The instrument is initially set-up as in B then sample is injected.

b. The mobile phase composition is changed to (acetonitrile:water) 60:40 and 70:30 then the sample is injected.

c. The best composition is determined by comparing the resolution of the chromatogram produce.

3. Identification of components in standard mixture

For identification of components in methyl esters mixture, each compound was

injected individually to identify the components of the mixture using the optimized LC

conditions.

4. Separation using gradient elution

Based on the separation above, a gradient elution separation was performed to improve

the efficiency of the column.

RESULT

Response Factor Area Mobile Phase Ratio Retention Time

(minute)

52985.5 5298550 50% H2O:50% ACN 0.538

145506.21 14550621 60% H2O:40% ACN 0.621

134896.63 13489663 70% H2O:30% ACN 0.667

Sample ID: Caffiene Standard (100 ppm)

Respond Factor (RF) = Peak Area

Sample Amount (ppm)

For Standard 1 = 5298550 = 52985.5

100

For Standard 2 = 14550621 = 145506.21

100

For Standard 3 = 13489663 = 134896.63

100

Respond Factor Area Mobile Phase Ratio Retention

Time

(minute)

180227.45 18022745 50% H2O : 50% ACN 1.902

547649.01 54764901 60% H2O : 40% ACN 3.114

1323032.63 132303263 70% H2O : 30% ACN 6.483

Sample ID: Methylbenzoate Standard (100 ppm)

Respond Factor (RF) = Peak Area

Sample Amount (ppm)

For Standard 1 = 18022745 = 180227.45

100

For Standard 2 = 54764901 = 547649.01

100

For Standard 3 = 132303263 = 1323032.63

100

Respond Factor Area Mobile Phase Ratio Retention Time

(minute)

353914.45 35391445 50% H2O : 50% ACN 3.281

1031182.33 103118233 60% H2O : 40% ACN 6.471

1272972.99 127297299 70% H2O : 30% ACN 15.316

Sample ID: Phenatole Standard (100 ppm)

Respond Factor (RF) = Peak Area

Sample Amount (ppm)

For Standard 1 = 35391445 = 353914.45

100

For Standard 2 = 103118233 = 1031182.33

100

For Standard 3 = 127297299 = 1272972.99 100

DISCUSSION

During this experiment, a High Performance Liquid Chromatography (HPLC) Agilent

G1314A equipped with UV detector, 5 mm Reverse Phase C18 column and 10 µl sample loop

was used. At flow rate 1.5 ml / min and detector wavelength at 254 nm, the mobile phase ratio

(v/v) was set at 50% water and 50% acetonitrile at the beginning in order to analyze and observe

the effect of mobile phase on LC separation. After all the standard samples which is phenatole,

methylbenzoate and caffiene were injected, the ratio was changed to 60%:40% and 70%:30%

respectively on the same mobile phase. By the actual procedure, from this experiment we need to

identify the components contained in the standard mixture by using the optimized LC conditions

getting from the above ratio of the mobile phase as well as we should perform a gradient elution

separation to improve the 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, increasing

amounts of water are added to acetonitrile to create a continuous gradient.

But what was happened is all the peaks from the injection process to the sample loop

were not separated well. In a reversed-phase separation, eluent strength decreases as the solvent

becomes more polar. Acetonitrile has high eluent strength, and all compounds are eluted rapidly.

All the peaks are observed overlapping. From the result of chromatogram and area calculation,

we can see that the Response Factor for all the standards injected is almost same. It was so

difficult to determine the resolution of the peaks since the peaks got overlap because the mixture

is in high concentration. As we know, the quantitative analysis in separation method depends

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

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

peak should be totally resolved from any neighboring peaks. A co-elution or other anomalies

such as tailing or fronting will distort or obscure the beginning and ending points of the peak.

There are some factors that contribute to all the problems stated above. The sample must

be degassing properly. Sometime when the pressure was not consistent, there must be any air

bubble in the mobile phase that fluctuant the instrument. Therefore the instrument should be

purge to let the pressure stable. Mobile phase that is too cooled also effect the pressure. The

254nm is the most suitable wavelength because give us very nice and sharp peak. The flow rate

or velocity of the mobile phase is very essential in HPLC (according to the Van Deemter

Equation).

CONCLUSION

In this experiment, students would know how the concept and method development of

optimizing a separation of a standard compounds using High Performance Liquid

Chromatography (HPLC) by varying the mobile phase composition instead of other factors that

effected to the elution process occurred.

REFERENCES

1. Skoog, Holler and Nierman, 5th Edition. Principles of Instrumental Analysis.

Thomson Learning 1998

2. Skoog, D.A., West, D.M, Holler, F.J. 7th Edition, Fundamental of Analytical

Chemistry