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Pmy2106 > Chromatography > Flashcards

Chromatography Flashcards

1
Q

Principle of hplc

A

Injection of a Smakk volume of liquid sample into a column packed with Tiny particles called stationary phase

Individual components of the sample are moved down packed column with the mobile phase forced through the column by a hugh pressure pump.

The components are separated by one another by the column packing that involves various Chemical or physical interactions between molecules and the packing particles.

The separated components are detected at the exit of the column by a detector that measures their amount. An out out from thus detector is called a liquid chromatography

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2
Q

Types of HPLC

A

Normal phase
Reverse phase
Adsorption
Ion chromatography
Size exclusion chromatography

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3
Q

Normal phase chromatography

A

This is not common.

It is the traditional separation mode based on the adsorption/ desorption of analyte onto a polar stationary phase (typically silica or alumina )

Polar analytes are going to migrate slowly through the column due to strong interactions with the polar stationary phase

It is also particularly useful for the separation of non polar compounds and isomers

One disadvantage us the easy contamination of surfaces by sample components

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4
Q

Reverse phase chromatography

A

This is more common

Based on analytes partition coefficient between a polar mobile phase and non polar stationary phase

Non polar analytes will interact more strongly with hydrophobic groups for informing a liquid like layed around the solid silica support.

Sutible for analysis of polar , medium polarity and some non polar analytes

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5
Q

Ion exchange chromatography

A

Based on the exchange of ionic analytes with counter ions of ionic groups attached to the solid support

Typical exchange groups are anionic (so3-) or cationic (nh4+) groups bonded to polymeric or silica materials

Common applications are the separation of ions and biological components such as amino acids

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6
Q

Size exclusion chromatography

A

Separation mode based on analytes moleculer size

Larger molecules are excluded fro, the pores and migrate faster than the smaller Molecules that can penetrate the pores

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7
Q

Instrumentation of hplc

A

See notes

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8
Q

What affects HPLC resukts

A

SAMPLE PARAMETERS
conc
Nature of sample eg biological
Sample extraction
Volume of injection

COLUMN PARAMETERS
column material type
Column material particle size
Column length dimsgees etc
Temp and pressure

MOBILE PHASE
Slovent
Ratio of solvent
Flow rate or pressure
PH

DETECTOR
wavelength
Sensitivity

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9
Q

How does mp need to be prepared

A

Needs to contain a buffer
Degas solvent as Small gas bubbles in the mobile phase can lead to collection in pump heads and detectors and ruin the analysis
Should be filtered to remove any particulate matter which could cause wear to the pumping system

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10
Q

What does mobile phase have an influence on

A

Solute retention and serparation

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11
Q

What is solvent strength refer to

A

It’s ability to elute solutes from a column

Increased solvent strength = faster elution

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12
Q

What is solvent strength related to

A

It’s polarity

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13
Q

In rpc what is a string and weak solvent

A

Strong - non polar hexane
Weak - water

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14
Q

Role of ph in mobile phase

A

If you ionise a drug by changing the ph it’s going to spend more time in the polar phase.

In rpc low ph is used to suppress the ionisation of weakly acidic analytics leading to a higher retention
Used a buffer eg acetic acid

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15
Q

% ionised

A

Sheet

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16
Q

How does flow rate and column temp affect mp

A

Operating at a higher flow rate Will reupducd retention and analysis time

Hugger column temps reduce the viscosity of mp and has effect in retention

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17
Q

Purpose of pump

A

Ensure delivery of precise, reproducible , constant and pulse free flow of mobile phase

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18
Q

What type of way can the pump deliver solvent

A

Constant mobile phase compition (isocratic )
Or increasing mobile phase composition (gradient )

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19
Q

Isocratic elution

A

The same mobile phase is used throughout the entire elution of the sample , mobile phase solvent remains constant with time
This is best for simple separations
Often used in quality control applications that support and are in close proximity to the manufacturing process

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20
Q

Gradient elution

A

The strength of the mobile phase in increasing with elution
Best for analysis of complex samples
Often used in method development for unknown liquids
Gives better resolution and faster elution

21
Q

Generation of gradient flow

A

LOW PRESSURE MIXING
The amount of each solvent (uo to four solvents ) is regulated by a proportioning valves . The mixed solvent then enter the hugh pressure pump and flows into the column

It is less expensive as it requires only one pump however gas bubbles are formed as the solvents are mixed at atmospheric pressure

HIGH PRESSURE MIXING
the delivery of multiple hugh pressure pumps is controlled individually with a programming deceive , and mixture is sent ti a hugh pressure mixing chamber.

Is expensive bc multiple required but a operate reliably without de gassing because the solvents are mixed at sufficndlty hugh pressure

22
Q

What is a HPLC column

A

Stainless steel tube filled with small particle packing

23
Q

Structure or silica particles surface

A
24
Q

What is retention time

A

The time between sample injection and peak Maximum (tR)

25
Q

Draw a graph with all measurements

A
26
Q

Where is peak width usally measured

A

At the base (wb) or at the peak half height (w1/2)

27
Q

What is vR

A

Retention volume. The volume of mobile phase needed to elute an analyte at a given flow rate.
VR= tRF

28
Q

What is VM

A

Void volume of liquid mobile phase needed contained in the column
Vm= tM F

29
Q

Peak volume

A

Also called band with is the volume of mobile phase containing the elute weak

Peak cpume = wbF

30
Q

How to calculate assymetry value

A
31
Q

How to calculate tailing factor

A
32
Q

What is resolution

A

Resolution (Rs) is a measure of the degree of separation of two separate analytes

33
Q

What does rs value need to be for baseline separation

A

1.5

34
Q

Now to work out resolution

A
35
Q

What Is a measure of column efficiency

A

Number of theoritical plates of plate number (N)

36
Q

How to work out number of theoretical plates

A
37
Q

HETP

A

Height of a theoritical plate = L/N
L= length of column

38
Q

What is the main factor controlling HETP

A

Particle diameter of the packing

39
Q

What number is h/ HETP usallly

A

2.5

40
Q

What is capacity factor

A

Capacity factor k’ is a measure of the degree of retention of solute by the column

It is the time the solute spends in the stationary phase tR’ relative to the time it resides in the mobile phase (tM)

41
Q

Capacity factor /retention factor eqn

A
42
Q

What do you use to establish peak identity

A

Retention time

43
Q

What do you use to measure conc

A

Band size

44
Q

How do you overcome errors reuniting from calibration curve when trying to calculate concentrations

A

Use an internal standard

45
Q

What is an internal standard

A

An exogenous compound that is added to both the calibrators and samples at the same time

46
Q

What determines the choice of is

A

Never be found in sample
Well resolved
Structural similar to analyte
Stable available in pure form

47
Q

How to work out conc

A

A series of solutions are prepared under varying concentrations of the compound under investigation plus a fuxed conc of internal standard.

Instead of dealing with area now , dealing with ratio of analyte response to is response

When you inject these solutions into the chromatogram obtained it will show a peak for the analyte sand is
From such traces yiu can calculate the peak area or height rations

Phr = huegh of sample peak / height if IS peak

Plot graph of peak height ration v conc of analyte
Get equation of lie and r2

48
Q

Advantages of hplc

A

Rapid and précise quatatuve analysis
Amendable to diverse samples
Automated operates
Can be used to quantify
Hugh sensitivity

49
Q

Limitations

A

No universal detector
Less seperation efficiency than capillary gc
More difficult for novices

Pmy2106 (14 decks)

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