Section 6 Analytical Method Development -Reversed phase HPLC for Neutral Analytes

Question 1

What phase is this?

Answer 1

Stationary Phases

Question 2

List the 4 different stationary phases?

Answer 2

Alkyl

Phenyl

Cyano

Silica

Question 3

Method Development Definition:

Answer 3

Selection of separation conditions that
provide an acceptable separation of a given sample in a minimum period
of time

Question 4

What are the Principles of HPLC Retention Mechanisms:

What order is reversed phase HPLC?

What are stationary phases environment?

What does the mobile phase typically consist of?

What is analytes retention govern by?

In london dispersion forces is thee some degree of retention?

Answer 4

1- The second chronological mode of chromatography

2- Stationary phases are hydrophobic

3- Mobile Phases typically consists of aqueous portion, which may or may not be pH adjusted, and an organic matter solvent

4- Analytes retention is governed by its HYDROPHOBICITY

Yes there is some degree of retention.

Question 5

What does stationary phases exist for?

Whats the most common phases?

How can altered selectivity be achieved?

Answer 5

Stationary phases exist for hydrophobic and hydrophilic analytes

The most common phases are the hydrophobic alkyl chains- C18, C8 and C4

Altered selectivity can be achieved using cyano, phenyl and amino phases where dipole - dipole and pi, pi hydrogen bonding interactions can be utilised.

Question 6

Retention Mechanisms :
Analyte Retention Reversed Phase HPLC:

What gives a good indicator as to its retentivity?

The less water soluble the more…

The more carbons present, the more…

The straighter the chain the more…

Full saturation =

Whats the general order of elution?

Answer 6

Chemical structure of the analyte gives a good indicator as to its retentivity

The less water soluble , the more retention

The more carbons present , the more retention

The straighter the chain (As opposed to branched), the more retention.

Full saturation , the more retention

The general order of elution =
Aliphatics > induced dipoles > permanent dipoles > weak bases > weak acids > strong acids

Question 7

If something has more carbons =

Answer 7

More polar

Question 8

Non polar=

Answer 8

More retention

Question 9

Polar =

Answer 9

Less retention

Question 10

List the retention order:

Answer 10

1 Straight chain hydrocarbon – most
hydrophobic (C11)

2. Straight chain hydrocarbon – less
   hydrophobic (C10)
3. Branched n-hydrocarbon (C10)
4. Unsaturated C10 - (more polar due to π
   electron dipole in the double bond)

5-7. Analytes with functional groups. Ionized
analytes will elute fastest of all

Question 11

Partition Coefficient (Log P)
Review of retention Mechanisms:

Is this useful?

What does it determine?

When is it typically determined?

What does Log P Value 1 favour?

What does Log P Value -1 favour?

Answer 11

Useful to determine how well an analyte will be retained on reverse phase method.

Determines a neutral analytes affinity for the stationary phase

Typically determined in water and octanol

Log P 1 favours - Organic portion

Log P -1 favours - Aqueous portion

Question 12

Whats the Log P calculation ?

Answer 12

The amount of analyte that’s in organic vs aqueous layers

Question 13

Assessment of sample
composition/instrumentation

List the 6 different ways to access:

Answer 13

Solubility (Log Pow)

Molecular Weight

pka – Acidic/Basic – positive/negative/neutral

UV absorbance (ƛ -max)

Stability –/light/pH/temperature

Degradation Pathways

Question 14

Assessment of sample
composition/instrumentation

Whats the most common way to access?

Answer 14

UV absorbance (ƛ -max)

Question 15

What is a silonal group?

Answer 15

Its a weak acid

Question 16

Mobile Phase used in Reversed Phase HPLC

What does the mobile phase consist of?

What is the weakest solvent and why?

What does the organic modifier do ?

Answer 16

Mobile phase generally consists of water and an organic solvent

Water is always the weakest solvent as it repels the hydrophobic analyte onto the stationary phase the most.

The organic modifier (strong) reduces the polarity of the mobile phase thus attracting the analyte back into the mobile phase

Question 17

If you increase the retention factor what do you also need to do?

Answer 17

Increase the water in Stationary phase and this increases the London dispersion forces

Question 18

What does more methanol or acteonele in the column do?

Answer 18

It pushes it down the mobile phase quicker

Question 18

What does more organic modifier compete with?

Answer 18

Competes with analyte and reduces interaction with the column stationary phase

Question 19

What does Synder Polarity Index Values show?

Answer 19

The strength of organic solvent

It sets up a perfect environment for London dispersion forces

Question 20

List the solvents used in reversed phase HPLC: (3) And there pros

Answer 20

Acetonitrile has lower viscosity - reduces back pressure and often results in slightly better peak shape

Acetonitrile has lower UV cut off - advantage for UV detection

Methanol is less expensive and less toxic

Methanol is more polar - reducing risks of solid buffer precipitation

Question 21

Whats the most common solvent for reversed phase in strength ?

Answer 21

Acetonitrile and Methanol

Acetonitrile = alot of sensitivity on stronger wave lengths

Question 22

Mobile phase :

If you change the organic modifier what may that do?

Why is retention altered?

Changes in selectivity occur due to :

Answer 22

Changing the organic modifier may alter selectivity and retention i.e. MeCN to MeOH

Retention is altered due to a change in overall mobile phase polarity

Changes in selectivity occur due to each solvents differing ability to undergo acid, basic and dipole interactions with analytes

Each solvent will interact differently with differing analyte moieties

Question 23

What is acidity a a measure of?

What is basicity B a measure of?

What is dipole character an indication of?

Answer 23

Acidity a is a measure of the solvents ability to act as a proton donor

Basicity B is a measure of solvents ability to act as a proton acceptor

Dipole character indicates the solvents ability to interact via dipole and polarizability

Question 24

Retention and Selectivity are altered by changing: 3 things

Answer 24

Stationary phase - Chain length and chemistry , inclusion of polar moieties, exposure of silanol surface, polar end capping reagents Mobile phase - organic solvent type, % organic, pH, buffers and other additives Temperature - especially with ionisable analytes

Question 25

Less organic = More organic =

Answer 25

Less organic = more retention More organic = less retention

Question 26

What are the comparison of ways that selectivity can be modified?

Answer 26

Molecules of similar size are eluted in order of decreasing polarity Effect of solvent strength (compare a and b) Effect of temperature (compare a and c) Decrease in stationary phase hydrophobicity decreases retention (compare d and a)

Question 27

By decreasing the mobile phase strength what happens? By increasing the strength of the mobile phase what happens?

Answer 27

By decreasing the mobile phase strength (the amount of organic modifier) the retention time increases The analyte is more strongly repelled onto the stationary phase By increasing the strength of the mobile phase, retention decreases as the analyte has higher affinity for the mobile phase

Question 28

What formula does this relate to?

Answer 28

Effect of solvent strength on retention It is a method development tool Log K = retention factor We need 2 log K values S stands for shape selectivity constant. (It is constant and usually 4 for small molecules.) S increases for larger molecules. Φ is the volume fraction of B solvent (0.01 x %B), kw is the extrapolated value of k for solute X with water as the mobile phase (for Φ =0) and S is a constant for a given solute when only Φ is varied. For “small” molecules, (100 to 500 Da), S is approx. = 4. An increase in Φ by 0.1 unit (+10 % B) will result in decrease in retention for all peaks in the sample by 10(0.1x4) or about 2.5 fold. Thus this suggests a systematic procedure for arriving at a satisfactory value of % B so that 1 ≤ k ≤ 10 (or any other desired range of k)

Question 29

What can we increase to get a better retention factor?

Answer 29

We can increase water. Suitable value of % B can be found by starting at approx 80 % and working down to get 1

Question 30

Often, solvent strength can have an effect on what? What should your retention factor be between?

Answer 30

Selectivity Compare a and b. An intermediate value of 45 % is optimum. Retention factor should be greater than 2 and less than 15

Question 31

know and look at these ***

Answer 31

The Eluotropic series

Question 32

The relative strength of common solvents can be compared using what?

Answer 32

the Eluotropic series.

Question 33

In the The Eluotropic series what solvents are listed?

Answer 33

Tetrahydrofuran Acetonitrile (MeCN) Methanol (MeOG) Water

Question 34

In the The Eluotropic series for the solvents listed, what are the figures related to them?

Answer 34

Question 35

Effect of solvent type on retention Which solvent is strongest? Effect of mobile phase type

Answer 35

Methanol is weaker than acetonitrile and this is weaker than THF. 40% MeCN has the strongest solvent so retention factors for all peaks have dropped

Question 36

Reversed phase (partition) chromatography What does this graph show?

Answer 36

It shows where they are the same elution strength

Question 37

Do isoeluotropic mobile phase compositions have the same elution strength or different? What can they often display?

Answer 37

Isoeluotropic mobile phase compositions have the same elution strength. Separations are produced in the same timeframe (retention factor of last peak) Isoeluotropic mobile phases can often display altered selectivity (improved resolution, different elution order..)

Question 38

If you want to test for selectivity what would you swap?

Answer 38

Swap solvents and keep elution strength the same

Question 39

what can isoeluotropic mobile phases often display?

Answer 39

Isoeluotropic mobile phases can often display altered selectivity

Question 40

Solvent selection should be made according to the two chromatographic properties which are:

Answer 40

● elution strength (absolute retention) ● selectivity (relative retention among solutes)

Question 41

What can give you a change in selectivity?

Answer 41

Switching between organic solvents

Question 42

Iso - Eluotropic Mobile Phases What does Iso- Eutrophic mean? Where do Iso - Eutrophic solvents elute ? What can you use to find Iso - Eluotropic solvents?

Answer 42

Iso - eluotrophic - same elution power Solvents elute analytes in the same time frame with different selectivity Can use an iso - eluogram (nomogram) to find iso - eluotrophic solvent compositions

Question 43

Just look at this and understand the graph : Solvent type selectivity

Answer 43

(a,b): ACN alone is unable to resolve this Sample (c,d) vary MeOH using nomograph to match (a,b) above (d) Has near baseline resolution (R=1.4 for peak 4/5) Increasing (d) to 37% MeOH results in R=1.5 (not shown here) R=1.5 is not ideal for a final method so then try THF. 15% THF results in R=2.2

Question 44

Temperature can affect retention and selectivity of a separation true or false?

Answer 44

True- but not really for neutral components

Question 45

Why can selectivity can be altered and when is it particularly prevalent ?

Answer 45

The retention factor can either increase or decrease depending on the analyte and, hence, selectivity can be altered, this is particularly prevalent with ionizable analytes as pKa is temperature dependent.

Question 46

An increase in temperature will allow for what? The ability to work at higher flow rates is attributed to what? The reduction in the effect of the ‘C’ term of the Van Deemter equation, due to what?

Answer 46

An increase in temperature will allow the ability to work at higher flow rates. The ability to work at higher flow rates is attributable to a reduction in viscosity of the mobile phase as the temperature is increased The reduction in the effect of the ‘C’ term of the Van Deemter equation, due to the improvement of mass transfer kinetics (i.e. the movement of analyte molecules in and out of the stationary phase pores).

Question 47

You can get changes in selectivity by changing what?

Answer 47

The retention factor

Question 48

Column temperature selectivity Be able to explain the correlation between temperature and column selectivity:

Answer 48

* Increase in temp by 1oC results in decrease in retention of 1-2% for each peak (usually) * Effect much more prevalent for ionisable analytes since pH is temp dependant. * Peaks A, B and C are more RELATIVELY retained at higher temps..ie they move to the back (later) of the chromatogram but there is an overall decrease. If we increase temperature the overall run will be quicker but there will be a 1-2% change in peak

Question 49

The problem of missing or hidden peaks can be addressed using what?

Answer 49

The problem of missing or hidden peaks can be addressed using an orthogonal separation: a separation with very different selectivity (i.e. different mobile phase/column) that is therefore likely to separate the two peaks that were overlapped in the primary method. When we are looking for hidden peaks we use orthogonal selectivity

Question 50

Pumps in HPLC What are the requirements of the ideal pump?

Answer 50

* Constant and reproducible flow * Pulse-free flow * Flow rate of 0.1-10 ml/min * Pressure up to 400 bar (5800 psi) * Inert

Question 51

Pumps in HPLC List them: (2)

Answer 51

Binary pump (high pressure mixing) Quaternary pump (low pressure mixing)

Question 52

Pumps in HPLC – Mixing mobile phases Tell me about the Binary pump Total flow? Advantages and Disadvantages?

Answer 52

Used for high pressure mixing Two pumps working in unison but each delivering a different volume fraction of the mobile phase Total flow = 1 ml/min 50% A, 50% B A = 0.5 ml/min B = 0.5 ml/min Advantages: High pressure mixing = more reproducible gradient profile, esp at extremes of flow or gradient composition, lowest gradient dwell volume (can deliver gradients more quickly) Disadvantages: More components (maintenance)

Question 53

Pumps in HPLC – Mixing mobile phases Tell me about the quaternary pump Total flow? Advantages and Disadvantages?

Answer 53

It is a low pressure mixing pump Low pressure multi-proportioning valve: Four solenoid controllers which open/close based on “duty cycle” of pump. If duty cycle = 1sec, then for 25:25:25:25 mixing, each valve opens for 0.25 sec Pump flow rate remains constant during gradient Advantages: Four channels = more flexibility, less maintenance, less expensive, Disadvantages: Less accurate mixing, prone to outgassing, needs vacuum degasser

Question 54

Pumps in HPLC – Mobile phase preparation What does filtration do?

Answer 54

Filtration: Protect pump, tubing and column (esp. sub-2 µm) Vacuum filtration also degasses !

Question 55

Pumps in HPLC – Mobile phase preparation What is degassing?

Answer 55

Mixing aqueous solvents and organic modifiers produces gas. Air bubbles in pumps cause pulsation. Air bubbles cause: *Ultra sonication *Helium sparging *Vacuum degasser

Question 56

What do air bubbles cause? (3)

Answer 56

*Ultra sonication *Helium sparging *Vacuum degasser

Question 57

Sample injection in HPLC Where is the injection valve located ? The injection of a sample at atmospheric pressure into the system, at high pressure, represents what? What's important about injection quantity: Explain how its completed:

Answer 57

Injection valve located after pump = high pressure side. The injection of a sample at atmospheric pressure into the system, at high pressure, represents a critical step in the chromatographic process Injected quantity must be accurate, precise and not cause changes in pressure or flow.

Question 58

Whats the effect of sample mass (concentration) When we overload the column what happens?

Answer 58

Retention time decreases Solution = dilution

Question 59

When there is a volume overload what does that mean ?

Answer 59

Volume overload = injection volume is too high L in mm, dc in mm, Vs in uL, dp in um, Vpo is the baseline volume of a peak As long as Vs < 0.4 Vp0 then the increase in peak width and loss in resolution will be less than 10 % (which is acceptable)

Question 60

Effect of sample solvent Where should samples be dissolved? What does Injection solvents stronger than mobile phase do? If the injection solvent is sufficiently weaker than the mobile phase, then what happens? Based on a change in k of 2.5-fold for a 10% decrease in % B, use an injection solvent at least ?? weaker than mobile phase

Answer 60

Dissolve sample in mobile phase (mobile phase A) for gradients OR a weaker solvent …! Injection solvents stronger than mobile phase “washes” sample down the column until it is fully diluted in the mobile phase. If the injection solvent is sufficiently weaker than the mobile phase. Then the sample will be concentrated at the head of the column. Based on a change in k of 2.5-fold for a 10% decrease in % B, use an injection solvent at least 10 weaker than mobile phase

Question 61

What is an in line filter ? Whats the two common ones? Why would you use it? How it it put onto the filter?

Answer 61

Contains a frit (0.5 um is most common but 2.0 is common place for UHPLC) - should be similar to or smaller than the frit porosity on your analytical column Two common designs : -Re-usable holder that can be disassembled to access the frit -Disposable unit that screws directly into the end of the column Traps stray particles that could be injected with your sample (particulate material in the sample or from wear and tear of pump seals or injector rotor) Used to extend column life. Can also be used in extreme pH's. It is mounted directly downstream from the autosampler Change in frit when system back pressure increases significantly (+25%)