Development & Optimization of HPLC Method Course ...

Development & Optimization of HPLC Method

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Shimadzu (Asia Pacific) Pte Ltd 1

Course Outline

• Procedure for method development

• Optimization of separation in reversed phase HPLC

1

Steps for HPLC method development 1. Information on sample, define separation goals

2. Need for special procedure, sample pretreatment, etc?

8. Validate method for release to routine laboratory

7a. Recover purified material 7b. Quantitative calibration 7c. Qualitative method

3. Choose detector and detector settings

6. Check for problems or requirement for special procedure

4. Choose LC method; preliminary run; estimate the best separation conditions

5. Optimize separation conditions

What is Known Before Starting

Important information concerning sample composition and properties Number of compounds present Chemical structure (functionality) of compounds Molecular weights of compounds pKa values of compounds UV spectra of compounds Concentration range of compounds in sample of interest Sample solubility More…

2

Separation Goals • Is the primary goal quantitative analysis, the detection of an (undesired) substance, the characterization of unknown sample components, or the isolation of purified material? • Is it necessary to resolve all sample components? • If quantitative analysis is requested, what levels of accuracy and precision are required? • For how many different sample matrices should the method be designed? • How many samples will be analyzed at one time? • What HPLC equipment and operator skills are present in the laboratory that will use the final method?

Sample Pretreatment

Samples come in various forms: • Solutions ready for injection • Solutions that require dilution, buffering, addition of an internal standard, or other volumetric manipulation • Solid that must first be dissolved or extracted • Samples that require sample pretreatment to remove interferences and/or protect the column or equipment from damage.

3

Sample Pretreatment • Removal of insoluble material Filtration Centrifuge (Precipitation) • Control of concentration Concentration Dilution • Extraction Liquid phase extraction Solid phase extraction • Derivatization for detection OPA, ADAM, FMOC… • Hydrolysis ……

Selection of Detectors

• UV-VIS

Ultraviolet / Visible detector

• PDA

Photodiode Array detector

• RF

Fluorescence detector

• CDD

Conductivity detector

• RID

Refractive Index detector

• ECD

Electrochemical detector

• ELSD

Evaporative light scattering detector

• MS

Mass spectrometer detector

4

Developing the Separation • Which chromatographic method is most promising for this particular sample? HPLC, GC, CE, SFC, TLC? • What separation mode? Reversed phase, normal phase or …. • What column? Types of columns, temperature … • What mobile phase? Isocratic or gradient Buffer and pH

Selecting an HPLC mode

• Method development for most samples is often begun with reversed phase mode. • Ion pair reversed phase and normal phase are 2nd choice. • Samples exhibiting any of the following characteristics will often require special consideration. Macromolecules enantiomers other isomers inorganic ions Biological samples

5

Preferred experimental conditions for the initial HPLC Separation

Separation Variable

Preferred Initial Choice

COLUMN Dimensions (length, I.d.) Particle size Stationary phase MOBILE PHASE Solvents A/B %-B Buffer (compound, pH, concentration) Additives (eg., ion-pair reagents, amines) Flow rate TEMPERATURE SAMPLE SIZE Volume Mass

15 , 25 cmL x 2 - 6 mmID, 3-5 µm C-8 or C-18 Water (or buffer) - ACN 80-100% 10-100 mM phosphate, pH 2-7 not used initially 0.2-2 ml/min 30-50 oC ≤ 50 ul ≤ 100 ug

Characteristics of primary HPLC methods Reversed phase mode Uses water / organic mobile phase Column: C-18(ODS),C-8, phenyl, trimethylsilyl(TMS), cyano

First choice for neutral or non ionized compounds that dissolve in water /organic mixtures

Ion pair reversed phase mode Uses water / organic mobile phase, a buffer to control pH, and an ion pair reagent Column: C-18(ODS),C-8, cyano

Good choice for ionic or ionizable compounds, especially bases or cations

Normal phase mode Uses mixtures of organic solvent as mobile phase Column: silica, amino, cyano, diol

Good second choice when reversed phase or ion pair reversed phase mode are ineffective. First choice for lipophilic samples that do not dissolve well in water / organic mixtures, and for mixtures of isomers and for preparative scale HPLC(in this case, silica is best)

6

Characteristics of secondary HPLC methods Ion exchange mode Uses aqueous mobile phase plus buffer for pH control

Good choice for separation of protein and nucleic acid sample and related compounds Separation of inorganic ions (ion chromatography) is also used.

Column: anion or cation exchange

Size exclusion chromatography Uses either aqueous (gel filtration) or organic (gel permeation) mobile phase Column: diol for gel filtration; polystyrene or silica for gel permeation

Good choice for separating high molecular weight samples such as proteins and synthetic polymers GPC is used for molecular weight distribution measurements.

Improving the separation

• Resolution value for complete separation of two neighboring peaks should be value of Rs>1.5. • Value of Rs>2 should be the goal during method development of simple mixtures. • In case of multi component sample, value of Rs>1 may be the separation goal because of difficulty.

7

Separation goals in HPLC method development Goal Resolution Separation time Quantitation Pressure Peak shape Solvent consumption

Comment Precise and rugged quantitative analysis requires Rs > 1.5 < 5-10 min is desirable for routine procedures RSD 10 - Long running time - Broad band, cause (1) low sensitivity (2) poor accuracy in quantitation

Rs

0 2 4 6 8 10

k’= 2 - 10 is preferable.

2- 10 1- 20 0 2 4 6 8 10 12 14 16 18 20

k’

17

Solvent Optimization 100% MeOH 0.1