ANALYTICAL SEPARATION METHOD CHM 510

EXPERIMENT 2: HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC): METHOD DEVELOPMENT

NAME : NUR HAZIMAH BINTI MAHAMAD POZI PARTNER: SITI NORMALIA BINTI SULAIMAN LECTURER: GROUP: ASB2Ac DATE OF EXPERIMENT: 9/10/2013 DATE OF SUBMISSION: 23/10/2013

(2012448048) (2012237674)

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. Its 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 Flow rate Mobile phase : 254 nm : 1.5ml/min : 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 145506.21 134896.63

5298550 14550621 13489663

50% H2O:50% ACN 60% H2O:40% ACN 70% H2O:30% ACN

0.538 0.621 0.667

Sample ID: Caffiene Standard (100 ppm)

Respond Factor (RF)

Peak Area Sample Amount (ppm)

For Standard 1

5298550 100

52985.5

For Standard 2

14550621 100

145506.21

For Standard 3

13489663 100

134896.63

Respond Factor

Area

Mobile Phase Ratio

Retention Time (minute)

180227.45 547649.01 1323032.63

18022745 54764901 132303263

50% H2O : 50% ACN 60% H2O : 40% ACN 70% H2O : 30% ACN

1.902 3.114 6.483

Sample ID: Methylbenzoate Standard (100 ppm)

Respond Factor (RF)

Peak Area Sample Amount (ppm)

For Standard 1

18022745 100

180227.45

For Standard 2

54764901 100

547649.01

For Standard 3

132303263 100

1323032.63

Respond Factor

Area

Mobile Phase Ratio

Retention Time (minute)

353914.45 1031182.33 1272972.99

35391445

50% H2O : 50% ACN

3.281 6.471 15.316

103118233 60% H2O : 40% ACN 127297299 70% H2O : 30% ACN Sample ID: Phenatole Standard (100 ppm)

Respond Factor (RF)

Peak Area Sample Amount (ppm)

For Standard 1

35391445 100

353914.45

For Standard 2

103118233 100

1031182.33

For Standard 3

127297299 100

1272972.99

DISCUSSION During this experiment, a High Performance Liquid Chromatography (HPLC) Agilent G1314A equipped with UV detector, 5 mm Reverse Phase C18 column and 10 l 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