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SMA006 ADVANCED SDEPARATION TECHNIQUE

Supervisor :- Dr. Bob Ardrey

Fast-HPLCUniversity of Huddersfield

Vishnu Shinde (U1073020)

14/1/2011


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4. Quantitative and qualitative analysis of the eluted substances.

High Pressure/Performance Liquid Chromatography (HPLC):-

In HPLC system pressure is applied to the column, forcing the mobile phase through at much

higher rate. The pressure is applied using pumping system. The action of pump is critical,since it must not pulsate and mix up the sample being separated in the solvent, causing it tolose resolution. Development of pump has proceeded quite quickly over the last severalyears, and now it is possible to achieve good resolution under the condition required for HPLC. (1)A proper operation of HPLC system is depend on knowledge of principle of chromatographic

process, as well as reasons behind the choice of component of chromatographic system suchas mobile phase, column and detectors. Instrumentation of HPLC is shown in Figure 1. To

pass the mobile phase through the column at typical flow rates of 0.5-2 ml/min a high pressure pump is required. Injection device is used to introduce the sample to be separated;the injection of sample is may be manual or automatic, prior to column. The mobile phase

passes with the sample components eluting from column through low volume cell which isusually in detector. (5)HPLC has resolving power of chromatographic column increases with column length andnumber of theoretical plates per unit length, although there are limit to the length of a columndue to the problem of peak broadening. As the number of theoretical plates is related tosurface area of the stationary phase it follows that the smaller the particle size of stationary

phase, the better the resolution. Unfortunately, the smaller particle size, the greater theresistance to eluant flow. (1)HPLC allows you to use very smaller particle size for packing

material of column which provides greater surface area for interaction between mobile phaseand stationary phase. This gives much better separation of the component of the mixture. (4)Depending on relative polarity of solvent and stationary phase, there are two type of variancein use in HPLC. (4)

Normal phase HPLC (4):-Column is made up of tiny silica particles and solvent is hexane (non-polar). The polar components in mixture will stick longer to polar silica. Then the non-polar component will

pass faster through the column.

Reversed phase HPLC (4):-In reversed phase HPLC the column size is same, but silica is modified by attaching longhydrocarbon chain to make it non-polar. In this polar solvent is used for example a mixture of alcohol and water such as methanol. This is most common form of HPLC.


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Figure 1:- Instrumentation of HPLC (3)

Types of HPLC: - (10)

1. Adsorption chromatography.

2. Ion-exchange chromatography.

3. Size exclusion chromatography.

Fast HPLC:-

Introduction:-

Fast HPLC is popular topic today. Higher throughput demand is constantly growing. Thedevelopment of packing material with small particles and combine with other instrument(which gives increased back pressure) results in high resolution by fast HPLC. This all givesincreased use of Fast HPLC. (9)

In fast HPLC mobile phase should be liquid in nature to separate the mixture. The stationary phase is of liquid or solid phase. Components are dissolved in solvent, this is the first step.Then under high pressure this solution flows through a chromatographic column. The


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stationary phase present in column separates the components of mixture. The resolution of chromatogram is important which depends on extent of interaction between stationary phaseand solute components. The fast HPLC gives high degree of versatility which is not found inother chromatographic technique. Fast HPLC can separate wide variety of chemical mixtures.

(10)Instrumentation:-The instrumentation of fast HPLC is almost similar to HPLC, but few changes are there toincrease the resolution and to minimise the time for analysis. The fast HPLC increase thespeed by using short columns by this change it can give same efficiency like standard HPLCwith 3 or 5 m particles. The packing material size is small in fast HPLC therefore it requirevery high back pressure and smaller the particle size higher will be resolution. (9)

Fast HPLC has following parts, (10)

1. Stationary phase (column).

2. Mobile Phase.

3. Pump.

4. Injector.

5. Detector.

6. Data system.

1. Stationary phase (column):-

Stationary phase in fast HPLC separates the components from solvent which is passedthrough it. Stationary phase is of porous or non-porous material. Porous materialincreases resolution of chromatogram and decreases the time required for analysis.The porous particles efficiency and performance are excellent for analyticalseparation of small molecules (11). The stationary phase of 125 or 250 mm columns

packed with porous material transferred to 33 mm columns packed with non-porous particles. This change in fast HPLC reduces analysis time by factor four to eight (12).Silica is the best example for porous and non-porous material. The smaller pore sizegives better surface area but larger pore size material gives better separationespecially for large molecule.

2. Mobile phase:-

It is the phase in which analyte is present and forced through stationary phase for separation. If the stationary phase is polar then the mobile phase will be non-polar and


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vice-versa. We must choose the mobile phase which is compatible with detector. Theother parameters like pore size, sample solubility, viscosity are also considered whilechoosing the mobile phase. (10)

3. Pump:-

It is used to give back pressure to analyte which lowers the time of analysis. In fastHPLC the pressure is about 15000 psi/1000 bar. It means we can analyse compoundhaving particle size 1.7 m.

4. Injector:-

There are various injection techniques for injecting the sample in fast HPLC. Use of injection valve is simplest method of injection the sample. But nowadaysautomatic sampling devices are using. Inthis technique sample is introduced byauto sampler and microprocessors. (10)

Common HPLC injection system

5. Detectors:-

There are so many detectors present in the market. 2998 photodiode (PDA) detector isone of them. This detector gives advanced optical detection for fast HPLC. It hasflexible sampling rates for normal and fast HPLC separations. It has superior linear range. Thermal management is good which helps for maximum baseline stability (13).The mass spectrometer can be used as detector in fast HPLC.

6. Data system:-

The obtained data is electronic so some sophisticated software is used to analyse it. Itis fully analysed by computer. The use of electronic data is for minimise the errorsand increase accuracy. (10)

These parts are as standard HPLC but there are several parameters we can change in HPLC toimprove selectivity, some are as follows (6):-

1. Change in particle size.

2. Switch to monolith.

3. Change in column temperature.


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4. Change in column pressure.

5. Change in column length.

Fast HPLC is different than HPLC; this can be achieved in two ways

1. Narrow bore column.

2. Smaller particles.

1. Narrow bore column (2):-

Faster separation can obtain on standard HPLC instrument by using narrower internaldiameters with shorter column. Columns are available commercially with ids of 4.6,3.2, 2.1 and 1 mm. These offers reduced solvent (mobile phase) consumption andincreased mass sensitivity. The effect of using narrow, short bore column on HPLCsystem will be broader peaks and decreased resolution. (2)

By using different column id the flow rate will change

Table 1:- Flow rates at different column ID

Column ID Flow rate4.6 mm 1 ml/min3.2 mm 0.5 ml/min2 mm 0.2 ml/min1 mm 0.05 ml/min

2. Smaller particles:-

The same mass size particles of analyte are injected into column of different diameter then elution of material is in higher concentration with smallest diameter.

As compare to a 4.6 mm ID column a 2 mm ID column is five time and a 1 mm IDcolumn 20 times more mass sensitive. (2)

Advantages: - (14)

1. It is an automated process which takes few minute to analyse the sample.

2. It gives the high resolution compare to other chromatographic techniques.

3. Without problems the tests are reproducible.


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4. It gives higher signal to noise ratio.

5. The columns are cheap.

Disadvantages: - (14)

1. It is costly instrument.

2. The operation of fast HPLC may be complex.

3. It has low sensitivity for some compounds.

Discussion:-Figure 2 shows chromatogram which is obtained by column. The top most chromatogram isresult of analysis on standard HPLC by using traditional column (4.6 mm ID) beforeoptimisation. The overall separation was good and whole process done in less than 1.5

minutes with baseline resolution of all peaks.

The lower chromatogram shows results when HPLC is optimized and the changes are, (7)

Decreasing the tubing ID between column, injector and pump.

Decreasing the volume of UV detector flow cell by replacing the standard flow cellwith semi-micro flow cell from 13 L to 5 L.

Narrower peak widths and increased resolution will get by these changes in HPLC. Thensystem dead volume has been reduced. (7)Table 2:- Peak width and resolution before and after optimization of HPLC system. (7)

Before Optimization After OptimizationPeak Number Peak Width Resolution Peak Width Resolution

1 0.059 min ----- 0.042 min ----2 0.066 min 1.76 0.046 min 2.603 0.072 min 1.96 0.057 min 2.864 0.085 min 2.06 0.076 min 2.73

5 0.103 min 1.89 0.099 min 2.35Figure 2:- chromatogram of flow cell before optimization and after optimization (7)


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Fast HPLC is comparable to UHPLC: - (8)The fast HPLC method uses short packed column with 2.7 m fused core silica particles.These particles give rapid chromatographic separation at very low pressure. The UHPLC(Ultra High Pressure Liquid Chromatography) is another technique of rapid chromatographywhich works under very high pressure resulting from stationary phase with sub2-m

particles. UHPLC gives better chromatographic resolution, sensitivity and speed over

conventional HPLC. The fast HPLC is newly developed technique comparable to UHPLC inchromatographic performance but it do not require expensive UHPLC instrumentation, notnew laboratory protocols.

Conclusion:-

From above data it is conclude that the Fast HPLC is the best technique for analysis thesamples because it reduces the time which is required for analysis of sample and it gives

better resolution than conventional HPLC. It is comparable with UPLC. UPLC has most of

same advantages like fast HPLC. UPLC needs more pressure than fast HPLC but the analysistime is less than fast HPLC.

References:-

1. Insrumental Methods of Chemical Analysis, Gurdeep R. Chatwal and Sham K.Anand, First edition (2005)

2. SMA0006 Advanced separation Technique, Handouts by Dr. Bob Ardrey.

3. http://elchem.kaist.ac.kr/vt/chem-ed/sep/lc/hplc.htm Date: 20-12-2010 time: 15:00


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4. http://www.chemguide.co.uk/analysis/chromatography/hplc.html Date: 25-12-2010time: 12:00

5. http://www.forumsci.co.il/HPLC/WEB-Pharm_Review/HPLC_pharma_WEB_15-2-10.html

Date: 5-1-2011 time: 20:00

6. http://www.chromedia.org/chromedia?waxtrapp=xgsorDsHqnOxmOlIEcCzBcHjH&subNav=kjjrmJsHqnOxmOlIEcCzBcHjHC Date: 8-1-2011 time: 17:00

7. Optimizing performance for fast HPLC analysis on short columns; by Liming Peng,Tivadar Farkas and Philip J. Koerner; Phenomenex Inc., Torrance, CA, USA.

8. http://www.justchromatography.com/hplc/hplc-fused-core Date: 9-1-2011 time: 13:00

9. Fast HPLC using phase optimized liquid chromatography, by S. Lamotte, R. Brindle,K. D. Bischoff, M. Gokhle, K. Narkahede; Bischoff Chromatography, Boblinger Str.23, 712299 Leonberg, Germany.

10. http://www.standardbase.com/tech/HPLC.pdf, Date: 13-01-2011, time: 17:19

11. http://www.chem.agilent.com/Library/applications/5990-6001EN.pdf, Date: 13-01-2011, time: 20:00

12. fast HPLC with non-porous packing materials in the pharmaceutical industry; M.Wieser, P. Malz, T. Arendt, S. Kuppers; Humboldt universitat zu Berlin; unter denLinden 6, 10117 Berlin, Germany.

13. http://www.labwrench.com/?equipment.view/equipmentNo/4079/Waters/2998-Photodiode-Array--PDA--Detector/, date: 14-01-2011, time: 12:00

14. http://www.ehow.com/list_5911530_disadvantages-advantages-hplc.html, date: 14-01-2011, time: 18:44

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