Hplc method development
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The document discusses analytical method development for HPLC. It notes that method development requires selecting requirements, instrumentation type, and why. Existing methods may be unreliable, expensive, or time-consuming, necessitating new method development. Key steps in development include defining goals, establishing sample preparation, selecting detector and mode of separation, performing preliminary separations, optimizing conditions, and validating the method. Method development is informed by factors like number of analytes, sample matrix, and analyte properties.
![Nonionic polar
Nonpolar Ionic
Water-insoluble Water-soluble
Increasing polarity
Adsorption
Partition
Ion
Exchange
[ NP
Partition]
[ RP
Partition]
Size Exclusion
[Gel Permeation] [Gel Filtration]
MolecularWeight
102
103
104
105
106
7](https://image.slidesharecdn.com/hplcmethoddevelopment-180526091632/85/Hplc-method-development-7-320.jpg)
Hplc method development
- 1. Prepared by - P.Sai Praveen Analytical department 1
- 2. ANALYTICAL METHOD DEVELOPMENT: • Method development usually requires selecting the method requirements and deciding on what type of instrumentation to utilize and why. 2
- 3. 1. A suitable method for particular analyte in the specific matrix is not available. 2. Existing method may be unreliable (have poor accuracy or precision) 3. Existing methods may be too expensive or time consuming. 3
- 4. 1.Define method goals and the type of chromatography 2.Establish sample preparation procedure 3.Select detector 4.Select mode of separation (Column and mobile phase) 5.Perform preliminary separations 6.Optimize conditions 7.Validate the method 4
- 5. How many analytes of interest will be present – this helps to make choice on efficiency and resolution of the method What will be the sample matrix – Is there a need to separate analytes from many matrix components & require special sample preparations steps (such as SPE) Is the method qualitative or quantitative? What is the concentration of the analyte (We may need to deal with a range of analyte concentrations) – Is there a need for high sensitivity separation & detection techniques Analyte / Sample matrix solubility & stability –May need to exclude certain HPLC techniques when dealing with unstable or sparingly soluble compounds 5
- 6. Goal Comment Resolution Precise and Rugged Quantitative analysis requires that Rs be greater than 1.5 (preferably greater than 2.0) Separation time < 5 – 10 min is desirable for routine procedures Quantitation ≤2% for assays, ≤ 5% for less demanding analyses, ≤ 15% for trace analyses Peak height Narrow peaks are desirable for large signal to noise ratios Solvent consumption Minimum mobile phase use per run is desirable 6
- 7. Nonionic polar Nonpolar Ionic Water-insoluble Water-soluble Increasing polarity Adsorption Partition Ion Exchange [ NP Partition] [ RP Partition] Size Exclusion [Gel Permeation] [Gel Filtration] MolecularWeight 102 103 104 105 106 7
- 8. Chemical structure(s) Acidic/Basic, pKa Molecular weight Stability – light (photo stability) & solvents (soln . stability) Solubility Concentration Matrix 8
- 9. Sample preparation is an essential part of HPLC analysis, to provide a reproducible and homogenous solution i.e., suitable for injection on to the column. The aim of sample preparation is A sample that : Is relatively free of particulates & interferences Should not damage the column Should be completely soluble in mobile phase & compatible with the intended HPLC method 9
- 10. Weighing and Volumetric dilution (through aid of sonication/homogenization/dissolution) Sample extraction (for solid and liquid samples) Filtration Centrifugation To remove interferences Solid Phase Extraction (SPE) Derivatization 10
- 11. Ultraviolet/Visible Absorbance (UV/Vis) Mass Spectrometer (MS) Refractive Index (RI) Evaporative Light Scattering (ELS) Fluorescence (FL) Electrochemical (EC) 11
- 12. Selection is Based on: – Chemical nature of analytes and potential interferences – Limit of detection required –Availability and/or cost of detector 12
- 13. Requirement: analyte must be UV active Choose detection wavelength that maximizes sensitivity & specificity Solvents used may cause slight shifts in UVmax from published values (2-5 nm) * Check absorbance of analyte in mobile phase Mobile phase solvents have UV cut-off points. Operating below cut-off point will Reduce sensitivity & Add to baseline noise Diode Array Detector (DAD) can monitor multiple wavelengths simultaneously 13
- 14. When: Analytes don’t have UV absorption Analyte concentrations are too low for UV absorption Sample interferences are important Structural information is required (back to MS) 14
- 15. Requirement: analyte must be ionizable Can discriminate between co-eluting peaks in selected ion mode Reduces resolution required For best sensitivity, work at pH where analytes are ionized Neutral to basic pH (7-9) for acids Acidic pH (3-4) for bases 15
- 16. Universal detector Monitors difference in the refractive index of the sample cell vs. the reference cell Non-selective Concentration dependent Sensitivity is typically 100x-1000x less than a UV/Vis detector Cannot be used with gradients 16
- 17. Universal detector Detector is mass dependent and non-selective Ideal for: High molecular weight compounds, Sugars and less volatile acids Amount of light scattering is related to the molecular mass of the analyte Can be used with gradient systems Solvents should be volatile for best results 17
- 18. Analyte must fluoresce Excite at one wavelength, measure the emission at a longer wavelength Up to 1000x more sensitive than UV/Vis High specificity Concentration dependent Operation similar to a UV/Vis detector 18
- 19. Requirement: Analytes can be oxidized or reduced by an electrical current More sensitive than fluorescence Not as selective as fluorescence (typically) Not compatible with gradient elution 19
- 20. Nature of the sample and its solubility (Polar or non polar) How do analytes of interest differ from other compounds in sample? Reversed phase is the most frequently used mode 20
- 21. Mobile phase is polar Stationary phase is less polar Major distinction between analytes is their hydrophobicity The sample should be soluble in water or a polar organic solvent (eg. methanol) Examples are C18 (ODS), C8 (Octyl), Butyl, Phenyl, Phenyl-hexyl, PFP etc. 21
- 23. Mobile phase is non-polar while Stationary phase is polar Generally used for separation of non polar compounds Sample should be soluble in a hydrophobic solvent Mobile phase is a mixture of organic solvents without water. Eg. Hexane, Methylene chloride, isopropanol, MTBE, Ethyl acetate Mobile phase additives like TEA (for basic compounds) and acetic/formic acid (for acidic compounds) can be used. Examples for Polar Stationary phase: Cyano, Silica, Amino and Diol. 23
- 24. When analytes are ionic or potentially ionic Mobile phase is typically an aqueous buffer Mobile phase strength is a function of ionic strength pH is critical SAX is Strong Anion Exchange (WAX = Weak) SCX is Strong Cation Exchange (WCX = Weak) Examples Separation of Inorganic Cations and Anions, Organic Acids and Bases, Amino Acids, Nucleotides, Catecholamines, Peptides, Antibiotics 24
- 25. When analytes are ionic or potentially ionic Mobile phase is composed of a buffer, an ion-pair reagent and a polar organic solvent Typical ion-pair reagents include – Alkyl sulfonates (heptane sulfonic acid, octane sulfonic acid) for bases – Quaternary amines (tetrapropyl ammonium, tetrabutylammonium chloride) for acids 25
- 26. Major distinction between the analytes in the mixture is their hydrodynamic volume Generally for molecular weights > 2000 Want to avoid partitioning The mobile phase should be a strong solvent for the sample Aqueous SEC is called Gel Filtration – for separation of Proteins and other bio molecules Organic SEC is called Gel Permeation (GPC) – for separation of Polymers 26
- 27. Large proportion of analytes are water soluble Wide range of stable stationary phases are available Simple mobile phases work for many applications (i.e. Water : Acetonitrile) Retention and Selectivity are altered by changing: Stationary Phase -chain length and chemistry, polar end capping reagents etc. Mobile Phase -organic solvent type, % organic, pH, buffers and other additives Temperature-especially with ionisable analytes 27
- 28. In partition chromatography, the mobile phase should be a moderate to poor solvent for the samples Produce a capacity factor of 1 to 10 (Preferably 2 to 5) For ion exchange and size exclusion the mobile phase should be a strong solvent for the sample The use of additives or modifiers can – enhance a separation – Improves peak shape – Alters selectivity 28
- 29. Water miscible Low viscosity Low UV cut-off Unreactive Most commonly used: – Acetonitrile – Methanol – Tetrahydrofuran (THF) 29
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- 32. Use gradient elution Mobile phase strength changes over time Weak mobile phase early in the gradient – k >2 for weakly retained analytes Strong mobile phase later in the gradient – k <10 for strongly retained analytes Initial scouting run : Use to estimate % organic for an appropriate elution The gradient elution must elute all strongly-retained compounds 32
- 33. Longer run time Column re-equilibration required after every analysis Requires a pump with at least two-solvent capability Not compatible with some forms of detection (RI, EC) More variables to control for reproducibility Delay volume (dwell volume) becomes important Delay volume: Volume of mobile phase contained in the HPLC system between pump(s) and column 33
- 34. Solvent selection and mobile phase composition Gradient shape Gradient steepness - controlled by the mobile phase starting and ending composition and the gradient time. Duration and position of isocratic conditions Flow rate 34
- 35. Keep it as simple as possible Be aware that delay volumes will vary from instrument to instrument Make sure post run equilibration time is adequate to return column to initial conditions Pre-mix mobile phase modifiers Pre-mix solvents with poor miscibility Avoid ion-pair gradients 35
- 36. The method of choosing an appropriate HPLC for a particular separation varies widely from using a ‘favourite’ column from the column store, the recommended column from a similar method or to a rigorous screening of columns based on a study of analyte and/or column classification characteristics. A column is chosen based on the Knowledge of sample The properties of column packing material. On the expectation of how its components will interact with the packing material. 36
- 37. Silica based packing materials are used in about 75% of all HPLC separations performed today due to the physical stability Surface can be chemically modified with variety of bonded phases High efficiency of silica based HPLC columns Silica based packings are compatible with water and almost all organic solvents Reverse Phase columns: C18, C8, Phenyl, Phenyl-hexyl, PFP etc. Most HPLC separations are performed on bonded phase HPLC columns Octadecyl silica (C18) columns are the most widely used bonded phase columns in the reverse phase mode. 37
- 38. Column Dimension - Effect on chromatography • Short (30-50mm) - short run times, low backpressure • Long (250-300mm) - higher resolution, long run times • Narrow (≤ 2.1mm) - higher detector sensitivity • Wide (10-22mm) - high sample loading 38
- 39. pKa and Mobile Phase pH pH is an important consideration in method development. At a pH close to the pKa, peak distortion results due to partial dissociation of a weak acid or base into its conjugate form. If the mobile phase pH is near the pKa, small changes in pH can make large changes in retention – not what is desired for a robust separation. 39
- 40. 1 suitable for LC-MS as ammonium acetate 40
- 41. Temperature affects retention and in some cases selectivity An increase in column temp. by 1°C will decrease retention by 1-2% Increasing temperature can decrease pressure by reducing mobile phase viscosity It is desirable to have column thermostat to maintain constant Retention Time and resolution during routine analysis 41
- 42. Retention factor tRi - t0 k = t0 k2 α = k1 Selectivity N = 16 2t Wi Efficiency Resolution tR1 tR2 t0 w1 w2 Minutes0 10 mVolts 42
- 43. The ultimate goal of chromatography is to resolve two or more compounds into separate peaks. Resolution (Rs) is defined by the distance between two peaks relative to the widths of the peaks t2 , t1 are the retention times of the two components, W2 , W1 are the corresponding widths at the bases of the peaks 43
- 44. Resolution is proportional to square root of N To double resolution, N would have to increase by a factor of 4 N can be increased with longer column or smaller particle size – R ∝ √N – N column length∝ – N 1/particle diameter (but limited by column pressure)∝ 44
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- 46. k is longer when the solute has higher affinity to stationary phase. Practical limitation on how much Rs can be increased by changing k Increasing k’ has increasingly smaller benefit to Rs, especially at k > 5 Increasing k wastes valuable analysis time and the chromatographic peak height will decrease as the bandwidth of the peaks increases. k is changed by altering mobile phase strength 46
- 47. α is the ability of the chromatographic system to chemically distinguish between sample components Changing α is the most effective way to increase resolution α can be altered over wide range without sacrificing time or higher pressure Adjust α by changing stationary phase or mobile phase solvents High α values indicate good separating power and a good separation between the apex of each peak. 47
- 48. Parameter Usage Organic solvent Changing to a different solvent (e.g. Methanol to Acetonitrile in reversed phase HPLC) will alter the selectivity Mobile phase pH Can alter the degree of ionization of some analytes – affecting their hydrophobicity Solvent strength and additives Can be adjusted to affect selectivity as well as retention (capacity) factor Stationary phase One of the most popular ways to alter the selectivity of a separation Temperature Can have an effect with certain analytes in reversed phase and Chiral HPLC 48
- 49. 1.Adjust k to optimum range (~2-5) 2. If not close to desired resolution, adjust selectivity by changing either mobile phase or stationary phase. Return to step 1 3. If close to desired resolution, increase N (if needed) by increasing column length or decreasing particle size 49
- 50. Peak shape is often the controlling factor when optimizing complex separations, especially when components are present in very different concentrations. A chromatographic peak should be symmetrical about its centre and said to follow Gaussian distribution. But in practice due to some factors, the peak is not symmetrical and shows tailing or fronting. FRONTINGTAILING 50
- 51. Fronting is due to saturation of stationary phase or operating at low k value. Fronting can be minimised by reducing the solute concentration of the sample or reducing the injection volume of the sample operating at k value of ~2-5 ensures good peak shape as well as ensuring that the sample partitions in the stationary phase long enough to achieve separation. 51
- 52. Tailing peaks create issues with resolution (undetected minor bands in peak tail), quantitation and reproducibility. Tailing is caused by 1) adsorptive effects 2) column packing 3) injection solvent effects Elimination of peak tailing should start during method development, when the selection of the correct column & instrument parameters is made. If tailing starts suddenly it can be an indication of an instrument or column problem, or even something as simple as overloading the column with a poor injection. 52
- 53. Broad peaks occur due to the more conc. of sample, large injection volume, column deterioration. Ghost peaks occur due to the contamination of the column, compound from earlier injections. Negative peaks occur if mobile phase absorbance is larger than sample absorbance. Peak doubling occurs due to the co-elution of interfering compound, column over load, channeling in column. Base line spikes occur due to the air bubbles in the mobile phase and/or detector, column deterioration. 53
- 54. Variable Impact on Selectivity (α) Easy to Change Robust /Easy to Control? Stationary Phase Large No Yes Organic Modifier Large Yes3 Yes % Organic Modifier Medium Yes Yes Eluent pH Large1 No No Eluent Ionic Strength Small No No Temperature Medium2 Yes Yes Gradient Slope Large Yes Yes Use of an Ion Pair reagent Medium2 No No 1 – for ionisable analytes 2-large when analyzing ionisable species 3-when using quaternary pumping systems 54
- 55. One of the most difficult aspects of method development is knowing when we have achieved a suitable set of conditions and chromatographic outcomes. It is useful to consider what the completed might ‘look like’ prior to embarking on method development. 55
- 56. Define goals Gather information Select mode Adjust k Adjust α Optimize N if needed Know when to quit 56
- 57. Practical HPLC method development – Second edition by Lloyd R. Snyder, Joseph J.Kirkland http://www.chromacademy.com/lms/sco2/Theory_Of_ HPLC_Chromatographic_Parameters.pdf http://www.chromacademy.com/essential_guide_webc ast/mobile_phase_optimization_strategies_for_reversed _phase_hplc/mobile_phase_optimization_strategies_for _reversed_phase_hplc.pdf 57
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Editor's Notes
- #43: The retention factor or capacity factor, describes how long the molecule was retained on a column. Theoretically, it is the ratio of the number of molecules in the mobile phase to the number of molecules in the stationary phase. Optimum Retention Factor k for isocratic HPLC is 2 &lt; k &lt; 20 , corresponding to retention time between 1.2 min and 13.2 min for 5 cm HPLC columns. The selectivity or separation factor evaluates how well two peaks are separated. α larger than 1.1 is normally required for analytical purpose. For an analytical method developed for PrepLC separation, we desire selectivity as big as possible. Efficiency/Plate number is a measure of how narrow peaks are in relation to how long the compound is retained. Plate number is a leading indicator of how much the column could be overloaded. Plate number less than two thousand is usually unacceptable according to FDA guideline. For an analytical method developed for PrepLC separation, we desire plate number as big as possible. Resolution is a comprehensive measure of separation by considering all retention factor, selectivity, and column efficiency. The purpose of our method optimization is to maximize resolution for peaks of interest.