Basic Guidelines - High Performance Liquid Chromatography (HPLC-VWD) - Method Development .

This is simple instruction based guideline for beginners

1. First, you need to know analytes structure, solubility and choose the detector based on analytes or your interest. Use VWD detecter if the analytes are uv active or else use RID, LSD and MSD etc., HPLC method development start with literature survey to get rough idea about development; start with solubility of the analytes, we can try with reverse phase if it is soluble in polar solvents; if it is not we should use normal phase chromatography. Mostly many organic drug molecules are polar or mid polar so we usually go for RP-HPLC

2. Selection of wavelength by PDA , not always wavelength max, based on related compounds, UV cut off of mobile phase to avoid matrix interference etc. if the analytes are not UV active then there is dervatisation method but good analytical chemist will not prefer this as even a very good organic chemist fails in reactions

3. Column selection (start with C18 inertsil and short column (100mm or 150 mm) if it is revesed phase or u can use GL Sciences – Inertsil ODS, waters Xbridge, Agilent Zorbax eclips columns or phenomenex Gemini etc)

4. Mobile Phase: Gradient setting, always develope the method by using gradient than isocratic as it has advatange of reduce run time & increase peak shape. Use buffer or modifier if the analytes are ionisable because ionisable compounds will show as two peaks or split peak as it is existing in two form (HA and A-); so if you buffered the mobile phase into basic or acidic the analyte will go to single form. mostly chemist will choose acidic pH (2-4) as the most column will hydrolyze well in basic condition

HA ------------> H+ + A-

0.1% TFA, H3PO4, H2SO4, 1-2 % Acetic acid are the simple modifiers to restrict the pH into acidic side to improve the peak shape and well known phosphate buffer 0f 10 - 100 mmol phosphate buffe, optimum is 30-50 mmol

Start with mobile phase composition of Water & Methanol or ACN according to solubility in 50:50 ratio; feed other parameter like wavelength, temperature, 1.0 mL/min flow rate. choose conc. of analyte to fall the y axis or response between 200-800 AU (keep the peak response below 1000mV = 1V or 1000 mAU= 1AU to keep the UV detector linearity)

If there are more than 10 molecule in particular product / sample go for gradient method for better resolution & short run time.you can start with simple gradient

Time(min)---------%A(Water or Buffer)-------------%B (Organic Modifier-ACN, MeOH)

0---------------------80-----------------------------20

25-------------------20------------------------------80

35-------------------20------------------------------80

40-------------------80-------------------------------20

50-------------------80-------------------------------20

always follow ICH parameter about Tailing factor, Capacity factor, Asymmetry, theoretical plates while developing any method; change mobile phase composition, column etc. if needed; once you get better result then for sharpness or to save time adjust flow rate & increase Temp.

If our desired peak is not retain on water /Acetonitrile/Methanol composition mobile phase then go for Buffer; Phosphate buffer is widely use buffer for its PKa value,easily available, gives more Hydrophobic effect, stable or non degradable for 2-3 days.

5. ...............Before starting any method development on HPLC Flush the system with water, check pressure, saturate column with mobile phase, check calibration status, lamp Hours.

Method Development is very challenging task so always be logical before any changes in any parameter, always keep data about whatever trials you have done during method development.

HPLC method Development 10- 100 gm sample is enough but for overall development, Its a tentative quantity.

After method development method validation must be done. use ICH Q2(R2) for method validation

http://www.ich.org/products/guidelines/quality/article/quality-guidelines.html