First chromatographic method described (as a non-instrumental method).

Since samples don’t need to be initially vaporized, potentially any compound can be assayed by this method.

Instrumental development lagged behind that of GC because of difficulties in creating a stable solvent flow.

The stationary phase is a solid. Separation is due to a series of adsorption/desorption steps.

We’ll be spending most of our time with instrumental LC methods.

However, column and planer (TLC) methods can also be used.

Silica and alumina are common stationary phases.

Both solute and solvent are attracted to the polar sites on the stationary phase.

If solutes have differing degrees of attraction to the phase, a separation is possible.

Separation is based on solute partitioning between two liquid phases - (relative solubility).

This approach comes closest to our countercurrent extraction model.

More highly retained species have a greater affinity (solubility) for the stationary phase - compared to the mobile phase (solvent)

Separation of solutes is based on differences in this relative solubility.

1

2 3

4

1

2

3

4

5

5

The elution order will be somewhat reversed but not exactly - other factors must also be considered.

The stationary phase has an ionically charged surface, opposite that of the eluents.

+ + -

-

-

For instrumental LC, weak exchange resins are typically used.

These are typically exchange groups bound to a support.

The traditional exchange resin beads would be crushed under normal HPLC conditions.

Separation is based on molecular size. Stationary phase is a material of controlled pore size. Also called gel permeation.

Columns can be obtained that will separate specific size ranges.

Larger species will elute first - they can’t pass through as many pores so their path is shorter.

Useful for determining size and size range for polymers, proteins, ...

50

,00

0

25

,00

0

5,0

00

This example shows three general classes of components.

The second has a much larger size distribution.

Optimum solvent strength or polarity can be obtained by mixing solvents.

0.3 0.4 0.5 0.6 0.7

AC

N

MeC

l 2

met

hano

l

100

50

50 10

10

0 100

0

Snyder extended the method of solvent blending for reverse phase.

He recognized that polarity is just one factor that you can play with

It relies on the use of 4 solvents in developing the optimum separation.

Polarity is just one factor. The other is solvent selectivity.

Rs = N 4

(! - 1) ( ) k’ 1 + k’

polarity term

selectivity term

proton acceptors

proton donors

large dipole

0.2

0.2 0.7

0.7

0.7 0.2

II

IV

VIII V

VI

VII

I

III

Not all solvents are truly usable.

Can’t be mixed at any proportion May interact chemically UV absorption or viscosity is too high Toxic, too flammable High vapor pressure Too expensive

methanol - acid acetonitrile - base tetrahydrofuran - large dipole water - polarity adjustment

All are low viscosity available in high purity UV transparent miscible in each other

2. Create blends using each of the other solvents and water that have the same solvent polarity.

3. Evaluate each solvent for improvements in peak shape or movement of selective peaks.

4. A mix of any of the blended solvents is then evaluated for optimum resolution.

41% ACN, 59% water k’ = 5

30% ACN, 70% water k’ = 10

21% MeOH, 79% water k’ = 10

11% ACN, 12% THF 77% water, k’ = 10

•  Unlike GC, variations in temperature have minimal effect on an LC separation.

•  However, variations in solvent polarity can greatly affect retention.

•  This can be accomplished by altering the solvent mix during an analysis.

Not all LC methods can make use of gradient elution

ion exchange - yes liquid-liquid - difficult bonded phase - yes size exclusion - no adsorption - yes

Starting solvent should have a polarity that adequately resolves the first few components.

Final polarity should adequately resolve the last few species in a timely manner.

Now play connect the polarity - attempt various blending rates to separate the remaining components.

a b

c d

e

f

a - gradient controller d - column/pre-column b - pump/dampening system e - detector c - sample introduction f - data output

Unlike GC equipment, many HPLC systems have a modular design - can simply add a new ‘box’ to change/extent capabilities.

There is also a wider range of how to do things like produce a flow or gradient.

We’ll cover some of the basic approaches.

All solvents should be ‘HPLC’ grade. This is a type of reagent grade material. It has been filtered using a 0.2 µm filter.

You can purchase it or produce it yourself.

Filtered solvent helps extend pump life by preventing scoring. It also reduces the chances of a column plugging.

All solvents should be degassed prior to use. This reduces the chances of bubbles being formed in the column or detector. Oxygen present at high pressure can also cause a problem.

Methods that can be used Displacement with a less soluble gas Applying a vacuum Heating the solvent.

Each type of system has its own advantages and disadvantages.

Is the solvent reservoir limited?

Does it produce pressure pulses?

Can a gradient be produced?

solvent outlet gas inlet

solvent

convection current baffle

a b

c

d

to column

a - syringe d - motor b - seal e - fill system c - gearing e

Another non-pulsating system with a limited reservoir. Stepper motor/gear system allows for very fine flow control.

g

f

e

d c

b

a

a - motor b - gear c - seal d - piston e - solvent in f - check valves g - solvent out

One of the most common type of systems.

Unlimited reservoir system but expensive.

Another problem is that it produces variable pressure - must reverse stroke to refill.

pump fill

Since the pump must spend at least a portion of its time filling, the is a pressure drop during this phase.

start of fill

start of pump

This effect must be minimized or your peaks will all have pulses in them.

That would greatly affect your sensitivity and detection limit

One approach is to have a more rapid fill cycle compared to the pump cycle.

This does not eliminate the problem but does reduce it.

One could also use two or more pumps working in tandem.

This is a more expensive option.

In-line metal coil system Reduces pulse to +/- 3% at 240 psig. Low cost system

Flow passes through tube - possible contamination Limited range - about +/- 50-100 psi.

tube is flattened

T type metal coil.

With this design, flow does not pass through the dampner.

It still has the previous limitations

pump column

adjustment screw

Allows the user to minimize pulsing under actual operational conditions.

Can reduce pulses to < 0.1 %

external pressure source

Pressure source can be a gas or a liquid

Reduces pulses to < 0.1% External pressure can be monitored and controlled by the system.

Most expensive approach but the best usually is.

•  With LC, temperature programming is not typically an option in dealing with homologous series.

•  Instead, we rely on altering the nature or polarity of the solvent - gradient elution.

•  The controller is the device that allow you to create the gradient program.

•  Gradients are produced based on the type of pumping system you have.

a b

pump controller

mixing tee

•  These can be a bit more complex than with GC systems.

•  If you attempted a manual syringe injection, expect to find the plunger shot into the ceiling - you might be working with pressures as high as 5000 psi.

•  A simple approach would be to stop the flow and inject manually - not too good.

A very common approach is the use of sampling valves and loops.

sample

vent

solvent

sample

vent

solvent

column

You must use ‘zero dead volume’ valves.

Manual and automated valve systems are available.

Major limitation is fixed sample size. The loop must be changed in order to alter sample size - does not require that the flow be stopped.

Automated syringes

check valve

syringe

This method allow for adjustment of sample size. The motor driven syringe can provide sufficient pressure to inject sample past the check valve.

•  A small column added between the injection system and the analytical column.

•  It helps prevent entry of materials that might want to stay on the column from your sample or solvent.

•  Used to extend column life

•  Should be the same packing as the analytical column.

HPLC has seen significant improvement over the last 10 years primarily due to improved column technology.

Packings are more uniform and smaller.

Phases are commonly chemically bound to the packing.

Packing methods have improved.

As packing size is decreased, efficiency and pressure requirements are increased.

Common diameters for analytical work diameter plates

10 µm 5000 5 µm 9000 3 µm 15,000

All are for a 15 cm x 4.6 mm id column

Today, most packing fall into four categories.

Silica or alumina

Bound phases on either alumina or silica.

Gels

Controlled-pore glass or silica

Strong cation - sulfonic acid group

Strong anionic - quarternary amine

Weak anion - primary amine

Weak cation - COOH

Gels - organic or aqueous based

Controlled-pore - silica or glass

Must be selected based on pressure requirements and size range required for your application.

A solute property detector.

Sample must exhibit absorption in UV/Vis range. Solvent must not absorb significantly at the measured wavelength.

Types Filter photometer - single " Variable wavelength Multiwavelength.

Dual photocell detector

fixed "#filter

Light source

dual flowcell

If the filter is replaced by a monochrometer, you end up with a variable wavelength UV/Vis system

photodiode array The photodiode array allow you to simultaneously monitor a range of " or obtain complete spectra.

Bulk property detector - general purpose.

Based on refraction of light as it passes from one media to another. Presence of a solute changes the refractive index of the solvent.

solvent only sample present

Waters design

mirror

split flow cell

detector

light source

adjustment control

Varian design

detector

flow cell

adjustment control

light source

Bulk property detector. Measures changes in polarity of the

liquid phase passing through the cell.

Measures conductivity of the solvent. Useful for solutions of ions

•  Most frequently applied of electrochemical detectors.

•  A known potential is applied across a set of electrodes - typically a glassy carbon type

•  Ability to oxidize or reduce a species can be measured.

•  Typically limited to working with a specific class of materials per analysis.

electrodes

Several electrodes and combinations can be used. Allows for some interesting data.