Hplc Method Development And Optimization Biology Essay

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Any new or better method for the analysis of an analyte can be developed basing on tailoring the existing analytical approaches and instruction. Developing a method generally involves proper selection of the method requirements and on the instrumentation type. The development of a HPLC method involves decision regarding the choice of mobile phase, column, detector and method of quantitation must be addressed.

After selecting the instrumentation, it is important to find out the chromatographic parameters for the analyte. The properties of analyte(s) are helpful to accomplish the approximate composition and pH of the mobile phase to select the column nature, wave length to be employed or mass/charge ratio to be scanned at for detection of the compound, the concentration range to be followed and choice of a suitable internal standard for quantification purpose etc. In the literature such information will be available about the analyte or related substances.

The next step is optimization and preliminary evaluation of the proposed method. Optimization criteria must be established with awareness of the goals usual to any new method. Initial analytical parameters of merit like sensitive (measured as response per amount injected), limit of quantitation, limit of detection, and linearity of calibration plots are to be established. It is crucial that method development should be done basing on the analytical standards that are authentic and well identified and characterized.

Optimization stage involves improvement of the initial sets of conditions that are evolved from the first stages of development in terms of peak shape, resolution, plate counts, capacity, peak asymmetry, elution time, limit of quantitation, detection limits and overall ability for quantifying the particular analyte. Next stage involves evaluation of the results obtained during optimization to meet the goals of the analysis set forth by the analytical figures of merit. Basing on the evaluation, it is revealed whether more optimization is needed to meet the method requirements.

In the optimization of the method, there should be good peak symmetry, maximum sensitivity, a wide linearity range, minimum detection, minimum quantitation levels and a high degree of accuracy and precision. The other potential optimization goals are baseline resolution of the analyte of interest from other sample components, on-line demonstration of purity, unique peak identification and interfacing of computerized data for routine sample analysis. Absolute quantitation should be simplified methods that require minimal handling and analysis time.

Method optimization can be done either by manual or computer driven approaches. In the manual approach one experimental condition is varied at a time, while keeping all others constant and the changes in response are evaluated. The variables include mobile or stationary phase composition, flow rate, detection wavelength, temperature and pH. This optimization approach is usually expensive time and consuming. However, by this method there is much better understanding of the principle involved and of the interactions of the variables. In computer-driven automated method development efficiency is optimized while experimental input is minimized. This approach can be applied to many types of methods. It significantly reduces the time of analysis, energy, and cost of analysis.


Classifying the sample

The first step in the method developments is to characterize the drug whether it is regular or special. The regular compounds are those that are neutral or ionic. The inorganic ions, bio-molecules, carbohydrates, isomers, enantiomers and synthetic polymers etc are called special compounds. The selection of initial conditions for regular compounds depends on the sample type. The general approach for the reverse phase chromatographic method development is based on the following considerations.

The regular samples like pharmaceuticals (either ionic or neutral) respond in predicable fashion to changes in solvent strength (%B) and type (e.g. acetonitrile or methanol) or temperature. A 10% decrease in %B increases retention by about three fold and selectivity usually changes as either %B or solvent type is varied. An increase in temperature causes a decrease in retention as well as changes in selectivity. It is possible to separate many regular samples just by varying solvent strength and type. Alternatively, varying solvent strength and temperature can separate many ionic samples and some non-ionic samples.

The choice of the initial column, mobile phase and temperature is quite important. The initial conditions for RP HPCL method developed are given in Table.

Separation variable

Preferred initial choice

Column packing

C8 or C18 column, less acidic silica columns; if temperatures > 50C are planned, more stable, sterically protected packings are preferred.

Column configuration

15 - 0.46 cm column, 5 µm particles.

Flow rate

2.0 mL / min

Mobile phase

Acetonitrile-water (neutral samples) or acetonitrile-buffer (ionic samples); buffer is 25-50 mM potassium phosphate at pH 2-3 (lower pH preferable if column is stable).


35 or 40 °C

Sample size

<50 µL; 50-100g.

The column and flow rate

To avoid problems from irreproducible sample retention during method development, it is important that columns be stable and reproducible. A C8 or C18 column made from specially purified less acidic silica and designed specifically for the separation of basic compounds is generally suitable for all samples and is strongly recommended. If temperature > 50o C are used at low pH, sterically protected bonded phase column packing are preferred. The column should provide reasonable resolution in initial experiments, short run times and an acceptable pressure drop for different mobile phases. A 5 µ, 150 - 4.6 mm column with a flow rate of 2 mL/ min is good for different mobile phases as initial choice. These condition provide reasonable plate number (N=8000), a run time of < 15 min for a capacity factor k < 20 and a maximum pressure drop < 2500 psi for any mobile phase made from mixtures of water, acetonitrile and / or methanol.

The mobile phase

The preferred organic solvent (B) for the mobile phase mixture is acetonitrile (ACN) because of its favorable UV transmittance and low viscosity. However, Methanol (MeOH) is a reasonable alternative. Amine modifiers like tetra hydro furan (THF) are less desirable because they may require longer column equilibrium times, which can be a problem in method development and routine use of the method. They may occasionally introduce additional problems like erratic base line and poor peak shape. However, some samples may require the use of amine modifiers when poor peak shapes or low plate number are encountered.

The pH of the mobile phase should be selected with two important considerations. A low pH that protonates column silanols and reduces their chromatographic activity is generally preferred .A low pH (<3) is usually quite different from the pKa of values of common acidic and basic functional groups. Therefore, at low pH the retention of these compounds will not be affected by small changes in pH and the reverse phase liquid chromatographic method will be more rugged. For columns that are stable at low pH, a pH of 2 to 2.5 is recommended. For less stable columns, a pH of 3 is a better choice.

Separation temperature

Mostly the temperature controllers operate best above ambient(>300C). higher temperature operation also gives lower operating pressures and higher plate numbers, because of decrease in mobile phase viscosity. A temperature of 35-400C is usually a good starting point. However ambient temperature is required if the method will be used in laboratories that lack column thermostating.

Sample size

Initially a 25-50µL injection (25-50µg) can be used for maximum detection sensitivity. Smaller injection volumes are required for column diameters of below 4.5mm and /or particals smaller than 5 µm. the sample should be dissolved initially in water 1mg/ml or dilute solution of acetonitrile in water. For the final method development stage, the best sample solvent is the mobile phase the samples which cannot be dissolved in water or in the mobile phase should be dissolved initially in either acetonitrile or methanol and then diluted with water or mobile phase before injection.

Equilibration of the column with the mobile phase

The analytical column is completely equilibrated with the mobile phase before injecting the sample for analysis and retention data are collected for interpretation. This is done for ensuring accurate retention data. Equilibration is required whenever the column, mobile phase or temperature is change during method development, usually by flow rate at least 10 column volumes of the new mobile phase before the first injection. Some mobile phases may require a much longer column equilibration time (e.g. mobile phases that contain THF amine modifiers such as triethylamine and tetrabutylamine and any ion pair reagent).

Column equilibration and reproducible data can be confirmed by first washing the column with at least 10 columns volumes of the new mobile phase and injecting the sample and then a second washing at least 5 column volumes of the new mobile phase and reinjection of the sample. If the column is equilibrates, the retention times should not change by more than 0.02 min between the two runs.

Column Performance

The following values are used to assess overall system performance.

Relative retention

Theoretical plates

Capacity factor


Peak tailing factor

Plates per meter

The chromatographic peak shape and plate number are calculated to assess column performance. The asymmetry factor as should fall between 0.9-10.5 and number of theoretical plates should be >4000 for a 15cm; 5µm column at a flow rate of 2 mL/min. The number of theoretical plates foe well packed HPCL columns under optimized test conditions is given in the Table.

Particle Diameter(µm)

Column Length(cm)

Plate number N




























Evaluating peak shape and plate number

The requirements for a given separation usually determine the type and configuration of the column to be used. There are different suppliers for a given type of column. These columns vary generally in performance. Therefore, certain information concerning column specifications and performance is needed for use in method development and their routine performance.

The column plate number (N) is an important characteristic of a column. N signifies the ability of the column to produce sharp, narrow peaks for achieving good resolution of band pairs with small α values. The table 2.2 shows the typical plate numbers (small, neutral sample molecules) for well packed HPLC column of various lengths and particle sizes. A15 or 25 cm column of 5 µ particle are preferred as a starting point for method development. This configuration provides a large enough N value for most separation and such column are quite reliable. A column which gives large N value can easily recognize closely over lapping peaks. Short columns of 3 µ particle are useful for carrying out very fast separation (<5min). But these columns are less used because they are more susceptible to sampling problems, more operators dependent and more affected by band-broadening.

Peak asymmetry and Peak tailing

Columns and experimental condition that provide symmetrical peaks always preferred. Peaks with poor symmetry can result in inaccurate plate number and resolution measurement, imprecise quantitation, degraded resolution and poor retention reproducibility.