High performance liquid chromatography 214 is the most widely used of all of the analytical separation techniques. The reasons for the popularity of the method is its sensitivity, ready adaptability to accurate quantitative determinations, suitability for separating non-volatile species or thermally fragile ones, wide spread applicability to substance that are of prime interest to industry, many fields of science and the public.
The applications of chromatography have grown explosively in the last fifty years owing not only to the development of several new types of chromatographic techniques but also to the growing need by scientist for better methods for characterizing complex mixtures.
General methodology for the development of new HPLC methods 215-228
HPLC method development follows the series of steps summarized below.
Information on sample, objective of separation.
Need for special HPLC procedure, sample pretreatment etc.
Choice of detector and detector settings.
Choosing LC method, preliminary run, estimation of best separation conditions.
Optimization of separation conditions.
Check for problems or requirement for special procedure.
a) Recovery of purified material b) Quantitative calibration
c) Qualitative method
Validate method for routine laboratory use.
A good method development strategy should require only as many experimental runs as are necessary to achieve the desired final result. Finally, method development should be simple as possible, yet it should allow the use of sophisticated tools such as computer modeling if these are available.
Before the beginning of method development, it is necessary to review what is known about the sample in order to define the goals of separation. The kinds of sample related information that can be important are summarized in Table-7.1.
Number of compounds present in the sample
Chemical structures of components
Molecular weights of compounds
PKa values of compounds
UV spectra of compounds
Concentration range of various compounds in samples of interest
The chemical composition of the sample can provide valuable clues for the best choice of initial conditions for an HPLC separation.
Objectives of separation
The objectives of HPLC separation need to be specified clearly include.
The use of HPLC to isolate purified sample components for spectral identification or quantitative analysis.
It may be necessary to separate all degradants or impurities from a product for reliable content assay.
In quantitative analysis, the required levels of accuracy and precision should be known (a precision of ± 1 to 2% is usually achievable).
Whether a single HPLC procedure is sufficient for raw material or one or more formulations and / or different procedures are desired for the analysis of formulations?
When the number of samples for analysis at one time is greater than 10, a run time of less than 20 min. will be oftenly important.
Knowledge on the desired HPLC equipment, experience and academic training the operators have.
Sample pretreatment and detection
Samples for analysis come in various forms such as:
Solutions ready for injections.
Solutions that require dilution, buffering, addition of an internal standard or other volumetric manipulation.
Solids that must first be dissolved or extracted.
Samples that require pretreatment to remove interference and/or protect the column or equipment from damage.
Most samples for HPLC analysis require weighing and / or volumetric dilution before injection. Best results are often obtained when the composition of the sample solvent is close to that of the mobile phase since this minimizes baseline upset and other problems.
Some samples require a partial separation ( pretreatment) prior to HPLC, because of need to remove interference, concentrate sample analytes or eliminate “column killer”. In many cases the development of an adequate sample pretreatment can be challenging than achieving a good HPLC separation.
The detector selected should sense all sample components of interest. Variable-wavelength ultraviolet (UV) detectors normally are the first choice, because of their convenience and applicability for most samples. For this reason information on the UV spectra can be an important aid for method development. When the UV response of the sample is inadequate, other detectors are available (flourescence, electrochemical, PDA etc.) or the sample can be derivatized for enhanced detection.
Developing the method for the separation
Selecting an HPLC method and initial conditions
If HPLC is chosen for the separation, the next step is to classify the sample as regular or special. Regular samples means typical mixtures of small molecules (<2000 Da) that can be separated using more or less standardized starting conditions. Exceptions or special samples are usually better separated with a different column and customized conditions as summarized in Table-8.2. Regular samples can be further classified as neutral or ionic. Table-8.3 summarizes the appropriate experimental conditions for the initial reversed phase separation of regular samples. Samples classified as ionic include acids, bases, amphoteric compounds and organic salts. If the sample is neutral, buffers or additives are generally not required in the mobile phase. Acidic or basic samples, usually require the addition of the buffer to the mobile phase. For basic or cationic samples, “less acidic” reverse phase columns are recommended (Table-8.4) and amine additives for the mobile phase may be beneficial. Using these conditions, the first exploratory run is carried out and then improved systematically as discussed below.
Handling of special sample
Detection is primary problems; use ion chromatography
Some isomers can be separated by reversed-phase HPLC and are then classified as regular samples; better separations of isomers are obtainable using either (1) normal-phase HPLC or (2) reversed-phase separations with cyclodextrin-silica columns.
These compounds require “chiral” conditions for their separations.
Several factors make samples or this kind “special”; molecular conformation, polar functionality and a wide range of hydrophobicity.
“Big” molecules require column packing with large pores (>> 10-nm diameters); in addition, biological molecules require special conditions as noted above.
Preferred experimental conditions for the initial HPLC separation
Preferred initial choice
Dimensions (length, ID)
15 x 0.46 cm
C8 or C18
Solvent A and B
Buffer (compound, pH, concentration)
25mM potassium phosphate 2.0
Additives (e.g., amine modifiers, ion pair reagents)
Do not use initially
< 100 mg
B : Polar solvent
3.5 mm particles are an alternative using a 7.5 cm column
For an initial isocratic run; an initial gradient run is preferred.
No buffer required for neutral samples; for pH<2.5, pH-stable columns are recommended.
Smaller values required for smaller-volume columns (e.g., 7.5×0.46-cm, 3.5-mm column).
Physical properties of silica supports for some C 18 columns
Pore diameter (nm)
Surface area (m2/g)
Zorbax Rx, SB, XDB
a N/A : Not available
On the basis of the initial exploratory run isocratic or gradient elution can be selected as most suitable. If typical reversed-phase conditions provide insufficient sample retention, suggesting the use of either ion pair on normal phase HPLC. Alternatively, the sample may be strongly retained with 100% acetonitrile as mobile phase, suggesting the use of non-aqueous reversed-phase (NARP) chromatography or normal phase HPLC. Some characteristics of reversed-phase and other HPLC methods are summarized below.
Characteristics of primary HPLC methods
Method / description/ columns
Uses water – organic mobile phase Columns: C18 (ODS), C8, phenyl, trimethylsilyl (TMS), Cyano
First choice for most samples, especially neutral or non-ionisable compounds that dissolve in water-organic mixtures
Uses water-organic mobile phase a buffer to control pH and an ion pair reagent. Column : C18, C8, cyano.
Acceptable choice for ionic or ionizable compounds, especially bases or cations.
Normal phase HPLC
Uses mixtures of organic solvents as mobile phase Columns: Cyano, diol, amino and silica.
Good second choice when reversed-phase or ion-pair HPLC is ineffective, first choice for lipophilic samples that do not dissolve well in water-organic mixtures, first choice for mixtures of isomers and for preparative-scale HPLC (silica best)
Getting started on method development
One approach is to use an isocratic mobile phase of some average solvent strength (e.g., 50%) organic solvent. A better alternative is to use a very strong mobile phase with (80-100% B), then reduce %B as necessary. The initial separation with 100%B results in rapid elution of the entire sample, but few groups will separate. Decreasing solvent strength shows the rapid separation of all components with a much longer run time, with a broadening of later bands and reduced detection sensitivity.
Improving the separation and repeatable separation
Generally the chromatographers will consider several aspects of the separation, as summarized in Table-8.6.
Objectives of separation in HPLC method development
Precise and rugged quantitative analysis requires that Rs be greater than 1.5.
< 5-10 min is desirable for routine procedures.
£ 2% (1 SD) for assays; £ 5% for less-demanding analysis; £15% for trace analysis.
< 150 bar is desirable, < 200 bar is usually essential (new column assumed)
Narrow peaks are desirable for large signal / noise ratios
Minimum mobile-phase use per run is desirable.
a Roughly in order of decreasing importance but may vary with analysis requirements.
Separation or resolution is a primary requirement in quantitative HPLC. The resolution (Rs) value should be maximum (Rs>1.5) favours maximum precision. Resolution usually degrades during the life of the column and can vary from day to day with minor fluctuations in separation conditions. Therefore, values of Rs = 2 or greater should be the goal during method development for simple mixtures. Such resolution will favour both improved assay precision and greater method ruggedness.
Some HPLC assays do not require base line separation of the compounds of interest (qualitative analysis). In such cases only enough separation of individual components is required to provide characteristic retention times for peak identification.
The time required for a separation (run time = retention time for base band) should be as short as possible and the total time spent on method development is reasonable (runtimes 5 to 10 minutes are desirable).
Conditions for the final HPLC method should be selected so that the operating pressure with a new column does not exceed 170 bar (2500 psi) and upper pressure limit below 2000 psi is desirable. There are two reasons for that pressure limit, despite the fact that most HPLC equipment can be operated at much higher pressures. First, during the life of a column, the back pressure may rise by a factor of as much as 2 due to the gradual plugging of the column by particular matter. Second, at lower pressures < 170 bars, pumps, sample values and especially auto samples operate much better, seals last longer, columns tend to plug less and system reliability is significantly improved. For these reasons, a target pressure of less than 50% of the maximum capability of the pump is desirable.
When dealing with more challenging samples or if the goals of separation are particularly stringent, a large number of method development runs may be required to achieve acceptable separation.
As the experimental runs described above are being carried out, it is important to confirm that each chromatogram can be repeated. When changing conditions (mobile phase, column, and temperature) between method development experiments, enough time must elapse for the column to come into equilibrium with a new mobile phase and temperature. Usually column equilibration is achieved after passage of 10 to 20 column volumes of the new mobile phase through the column. However, this should be confirmed by carrying out a repeat experiment under the same conditions. When constant retention times are observed in two such back-to-back repeat experiments (± 0.5% or better), it can be assumed that the column is equilibrated and the experiments are repeatable.
Completing the HPLC method development
The final procedure should meet all the objectives that were defined at the beginning of method development. The method should also be robust in routine operation and usable by all laboratories and personnel for which it is intended.
Quantitation and method validation
One of the strengths of HPLC is that is an excellent quantitative analytical technique. HPLC can be used for the quantitation of the primary or major component of a sample (including pure samples) for mixture of many compounds at intermediate concentrations and for the assessment of trace impurity concentrations in matrix. Method validation, according to the United States Pharmacopoeia (USP), is performed to ensure that an analytical methodology is accurate, specific, reproducible and rugged over the specified range that an analyte will be analysed. Method validation provides an assurance of reliability during normal use and is sometimes described as the process of providing documented evidence that the method does what it is intended to do. According to USP, the method validation involves eight steps as given below.
Limit of detection
Limit of quantitation
Linearity and range
Precision and accuracy: Already discussed in chapter-1.
The linearity of the method is a measure of how well a calibration plot of response v/s concentration approximates a straight line, or how well the data fit to the linear equation. Y = aX + b
Where ‘Y’ is the response, ‘X’ is the concentration, ‘a’ is the slope and ‘b’ is the intercept of a line fit to the data. Ideally, a linear relationship is preferred (b = 0) because it is more precise, easier for calculations and can be defined with fewer standards. Also, UV detector response for a dilute sample is expected to follow Beer’s law and be linear. Therefore, a linear calibration gives evidence that the system is performing properly throughout the concentration range of interest.
Generally in HPLC, if we are using internal standard, then the linearity plot is to be drawn by taking concentration of the analyte on x-axis and the ratio of area under the curve (AUC) of analyte to AUC of internal standard (IS) on y-axis. The resulting plot slope, intercept and correlation coefficient provide the desired information on linearity. A linearity correlation coefficient above 0.999 is acceptable for most methods.
Limit of detection (LOD) The limit of detection (LOD) is the smallest concentration that can be detected reliably. The LOD represents the concentration of analyte that would yield a signal-to-noise (S/N) ratio of 3.
Limit of quantitation (LOQ) The LOQ is the concentration that can be quantitated reliably with a specified level of accuracy and precision. The LOQ represents the concentration of analyte that would yield a signal-to-noise ratio of 10.
LOD and LOQ can be determined by using the following expressions. LOD = 3 X N / B LOQ = 10 X N / B
Where N is the noise estimate, is the standard deviation of the peak area ratio of analyte to IS (5 injections) of the drugs.
B is the slope of the corresponding calibration curve.
The LOD and LOQ values determined during method validation are affected by the separation conditions, columns, reagents and especially instrumentation and data systems.
Method ruggedness is defined as the reproducibility of results when the method is performed under actual use conditions. This includes different analysts, laboratories, columns, instruments, sources, chemicals, solvents etc. method ruggedness may not be known when a method is first developed, but insight is obtained during subsequent use of that method.
The concept of robustness of an analytical procedure has been defined by the ICH as “ a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters”. The robustness of a method is the ability to remain unaffected by small changes in parameters such as pH of the mobile phase, temperature, percentage of organic solvent and buffer concentration etc. to determine robustness of the method experimental conditions were purposely altered and chromatographic characteristics were evaluated.
To study the pH effect on the retention (K1) of the drug, buffer pH is to be changed by 0.2 units. At certain point, retention will increase at any pH above and below of the pH unit.
The effect of temperature on the retention characteristics (K1) of the drug is to be studied by changing the temperature in steps 2ºC from room temperature to 80ºC and see the effect of temperature on the resolution and peak shape. Effect of percentage organic strength on retention is to be studied by varying the percentage of organic solvents like acetonitrile, methanol etc. from 0 to 2% while the other mobile phase contents are held constant and observe the K1. At certain point decreases in K1 observed with increase in the level of organic solvent. Effect of buffer concentration should be checked at three concentration levels i.e. 0.025 M, 0.05 M and 0.1 M and observe retention time and resolution.
To generate reproducible and reliable results, the samples, standards and reagents used for the HPLC method must be stable for a reasonable time (e.g., One day, one week, one month, depending on the need). For example, the analysis of even a single sample may require 10 or more chromatographic runs to determine system suitability, including standard concentrations to create a working analytical curve and duplicate or triplicate injections of the sample to be assayed. Therefore, a few hours of standard and sample solution stability can be required even for a short (10 min.) separation. When more than one sample is analyzed, automated, over night runs often are performed for better laboratory efficiency. Typically, 24 hours stability is desired for all solutions and reagents that need to be prepared for each analysis.
Mobile phases should be chosen to avoid stability problems, especially the use of amine additives or specific solvents. For example, mobile phase containing THF (tetra hydrofuran) are known to be susceptible to oxidation, therefore, the mobile phase should be prepared daily with fresh THF. Some buffered mobile phases cause problems for example, phosphate and acetate provide good media for microbial growth. Sodium oxide (0.1%) is often added to the mobile phase buffer to inhibit such growth, adding more than 5% of organic solvent is also effective.
Long term column stability is critical for method ruggedness. Even the best HPLC column will eventually degrade and lose its initial performance, often as a function of the number of samples injected.
System suitability experiments can be defined as tests to ensure that the method can generate results of acceptable accuracy and precision. The requirements for system suitability are usually developed after method development and validation have been completed.
The criteria selected will be based on the actual performance of the method as determined during its validation. For example, if sample retention times forms part of the system suitability criteria, their variation (SD) during validation can be determined, system suitability might then require that retention times fall within a ±3 SD range during routine performance of the method.
The USP (2000) defines parameters that can be used to determine system suitability prior to analysis. These parameters include plate number (N), tailing factor, k and / or a, resolution (Rs) and relative standard deviation (RSD) of peak height or peak area for respective injections. The RSD of peak height or area of five injections of standard solution is normally accepted as one of the standard criteria. For an assay method of a major component, the RSD should typically be less than 1% for these five respective injections.
The plate number and / or tailing factor are used if the run contains only one peak. For chromatographic separations with more than one peak, such as an internal standard assay or an impurity method, expected to contain many peaks, some measure of separations such as Rs is recommended. Reproducibility of tR or k value for a specific compound also defines system performance.
The column performance can be defined in terms of column plate number ‘N’ is defined by N = 5.54 (tR / W½)2
Where ‘tR’ is the retention time of the peak and ‘W½’ is the width of the peak at half peak height.
The resolution of two adjacent peaks can be calculated by using the formula Rs = 1.18 (t2-t1) / W0.5.1 +W0.5.2
Where ‘t1’ and ‘t2’ are retention times of the adjacent peaks and W0.5.1 and W0.5.2 are the width of the peaks at half height. Rs = 2.0 or greater is a desirable target for method development.
The retention factor k is given by the equation. k = (tR – t0) / t0 where ‘tR’ is the band retention time and t0 is the column dead time.
The peak symmetry can be represented in terms of peak asymmetry factor and peak tailing factor, which can be calculated by using the following formula. Peak asymmetry factor = B /A
Where ‘B’ is the distance at 50% peak height between leading edge to the perpendicular drawn from the peak maxima and ‘A’ is the width of the peak at half height.
According to USP (2000) peak tailing factor can be calculated by using the formula T = W0.05 / 2f
Where “W0.05” is the width of the peak at 5% height and “f” is the distance from the peak maximum to the leading edge of the peak, the distance being measured at a point 50% of the peak height from the base line.