Introducing 

Prezi AI.

Your new presentation assistant.

Refine, enhance, and tailor your content, source relevant images, and edit visuals quicker than ever before.

Loading…
Transcript

LEGEND

HPLC Method Development

Step

- Leanne Kwok -

Decision

Path

STEP 0

Part 1

Know your compounds:

pKa, acidic and basic functional groups

STEP 0

Part 2

Select a column type:

What stationary phase do you want to have?

STEP 1

Choose a method:

OR

Isocratic

(ISO)

100%B run

Gradient

(GRA)

Scouting gradient

Advantage of scouting gradient over isocratic 100%B:

- gives more information (ideal %B) in only one run compared to isocratic which may need two runs to get the same information

STEP 1

GRA

ISO

Scounting gradient

100%B

Organic range usually 5-100%B

tg usually 20 min

To ensure that all compounds are eluted with in 1-3 min

A 'potato' peak around this time

?

YES to

all

Run time < 20 min

K < 20

Rs > 1.5

NEARLY

All but Rs:

1.4 < Rs < 1.5

NEARLY

Run time < 20 min

K < 20

1.4 < Rs < 1.5

NO

to

all

STEP 2

ISO

Method change?

Decrease %B

ISO or GRA

To change to isocratic method a condition must be met: delta tg > 0.25xtg

Decreasing %B will increase run time and hopefully separate the 'potato' peak (from 100%B).

Can decrease B by 10% less (i.e. 100 - 10 = 90%)

This will help in calculation of multiplying factor, so we can then use the K Rule to help achieve goal of run time < 20 min and K < 20

Calculation:

RUN TIME and K GOAL

?

delta tg > 0.25xtg?

YES to

all

Run time < 20 min

K < 20

Backpressure < 4000 psi

Rs > 1.5

STEP 2

GRA

GRA -> ISO

NEARLY

All but Rs:

1.4 < Rs < 1.5

NO

to

all

Calculate %B

(start at 5-10% less than %B calculated)

Scouting gradient allows you to calculate a %B that can best be used to start isocratic method

Calculate %B as the tR for the first peak after t0 peak

STEP 3

Calculate multiplying factor

Multiplying factor (MF) = k2/k1 (shown in table -> (v) column 5, row 2)

Calculate %B for desired k value:

Example calculations:

slope: (organic range / tg)

= (100%-5%/20)

= 95%/20

= 4.75% per min

tR (first peak after t0) - t0

= 19.16 - 2 = 17.16 min

tR x t0 + (%B organic range start at)

= (17.16 min x 4.75% per min)+5%

= 81.51%+5%

= 86.51%B

(start at 5-10% less than %B calculated)

Run isocratic run at new %B

Run at newly calculated %B

?

Change organic range to start at new %B and end at 100%

Run at new organic range

NO

to

all

Run time < 20 min

K < 20

Rs > 1.5

NEARLY

All but Rs:

1.4 < Rs < 1.5

YES to

all

?

YES to

all

Run time < 20 min

K < 20

Backpressure < 4000 psi

Rs > 1.5

RESOLUTION GOAL

STEP 3

ISO

NEARLY

All but Rs:

1.4 < Rs < 1.5

NO

to

all

Change selectivity

There are a few approaches to changing selectivity:

50%ACN

-> Changing solvent:

ACN, MeOH

Mix (preferably done after the two solvents have been tried separately)

NOTE: remember to consult nomogram

STEP 3

GRA

65%MeOH

Changing selectivity

There are many approaches to changing selectivity:

Mix

Organic range, tg, flow rate, l

SELECTIVITY

-> Changing temperature:

from ambient to 50 deg C

SELECTIVITY

50 deg C

-> pH:

can separate two close peaks by their pka

pH 7

?

YES to

all

Run time < 20 min

K < 20

Backpressure < 4000 psi

Rs > 1.5

?

YES to

all

Run time < 20 min

K < 20

Backpressure < 4000 psi

Rs > 1.5

NEARLY

All but Rs:

1.4 < Rs < 1.5

NO

to

all

NEARLY

All but Rs:

1.4 < Rs < 1.5

NO

to

all

STEP 5

ISO

Change stationary phase

(Step 0 Part 2)

Change efficiency

There are many approaches to changing efficiency: flow rate, l, d, ps, pore size

Flow rate

When changing flow rate, may change %B as well to ensure run time and K are within limits

Change column:

l (250 mm, 150 mm, 100 mm)

d (4.6 um, 3.2 um)

ps (5 microns, 3 microns)

pore size (coreshell)

UPLC for smaller l, d and ps

EFFICIENCY

?

YES to

all

Run time < 20 min

K < 20

Backpressure < 4000 psi

Rs > 1.5

NO

to

all

Change stationary phase

(Step 0 Part 2)

Last Step

Method Validation

The purpose of completing a method validation is to ensure that the HPLC method was suitable.

METHOD VALIDATION

Precision

Accuracy

Limit of Detection

Limit of Quantitation

Specificity

Linearity

Range

Robustness

YES

NO

to

all

?

YES to

all

Run time < 20 min

K < 20

Rs > 1.5

NEARLY

Run time < 20 min

K < 20

1.4 < Rs < 1.5

NO

to

all

NO

YES to

all

Learn more about creating dynamic, engaging presentations with Prezi