MAAC: Chrompatography 5

Question 1

Describe how the instumentation works in HPLC

Answer 1

It is sometimes loosely referred to as high pressure liquid chromatography, this is because it requires really high pressure to push the liquid mobile phase through the column (commonly 3000psi).

In HPLC, the sample mixture (in liquid form) is filled into an injection loop, (of fixed volume) using a syringe. This loop is then switched by a valve into the flow of the mobile phase which takes it onto the column. This means the loop is filled up at room pressure and only subjected to column pressure when the valve is switched into-line.

The sample is carried onto and through the column by a continuous flow of liquid which acts as the MP. This liquid is delivered by a high-pressure pump(s).

Components in the sample separate according to their polarity and/or solubility

A detector located at the end of the column monitors the components as they elute from the column and forms the chromatogram.

Question 2

Describe Reverse phase and normal phase chromatography and give examples of SP and MP used in each

Answer 2

The chemical nature of the stationary phase within the column dictates the mode of HPLC.

Normal phase HPLC describes a system that has a polar stationary phase (silica particles) and a less polar mobile phase (e.g. hexane and cyclohexane).

Reverse phase HPLC describes a system which has a non-polar stationary phase (e.g. C8 octylsilica, C18 octadecyl silica or a phenyl packing) and a more polar mobile phase (e.g. methanol and water).

Over 90% of HPLC methods for pharmaceutical analysis employ reverse phase rather than normal phase HPLC.

Question 3

How is the elution of analytes effcted by the polarities of MP and SP?

How can the elution of analytes be manipultaed?

Answer 3

Stationary phases in reverse phase HPLC are non-polar, thus non-polar analytes will retain longest.

Given a mixture of analytes injected onto a reverse phase system the most polar analytes will elute first and the least polar last.

To allow manipulation of the elution of the analytes you can vary the polarity of the mobile phase being pushed through by the pump.

If you were to use a polar MP such as water, then very few of the analytes would elute.

If you were to use a less polar MP such as methanol then most of the analytes would elute.

It is by intelligent manipulation of the composition of solvents in the mobile phase that you can separate and elute all analytes from the column.

Question 4

1. What does high elution power or strength mean?
2. What does low elution power or strength mean?
3. What is the order for common solvents?

Answer 4

1. If a solvent use results in high analyte elution then we say it has a high elution power or strength
2. If a solvent use results in low analyte elution then we say it has low elution power or strength
3. In REVERSE PHASE CHROMATOGRAPHY the commonly used solvents are ranked in the following order:-

Water: methanol: acetonitrile: tetrahydrofuran

(—– Increasing elution power —->)

Question 5

Answer the following question

Answer 5

Longer HC chain on bottom one so top one eluted first

Question 6

Answer the following questions

Answer 6

Middle then first then last.

Look at additional methyl groups in each

Question 7

Answer the following quesions

Answer 7

Question 8

What are common methods that can be used to manipulate seperation of analytes in HPLC?

Answer 8

• Change the composition of the mobile phase:
  
  • Increase organic modifier to decrease overall retention
    
    • If retained too long on SP
  • Increase water content to increase overall retention
    
    • If not retained on SP long enough for seperation to occur
• Change the polarity of the column
  
  • Decrease from C18 to C8
  • Increase from C8 to C18

Question 9

What are less common methods used to manipulate seperation

Answer 9

Less common and more complicated:-

• Heating the column (isothermal increases)
  
  • More sol in MP
• Ion suppression
  
  • Change pH
• Ion pairing
• Gradient elution
• Derivatisation

Question 10

Look at the following slide.

It is important that analysis is optimised - Short time but maintain resolution of peaks. Look at the improvement on the use of gradient elution

1. What does Isocratic mean?

Answer 10

Maintain same MP composition over analysis run

Question 11

What instrumentation is required for gradient elution?

What happens to the peaks and time if gradient elution is used?

Answer 11

To achieve the change in organic modifier content with time, two or more pumps, a mixing chamber and a computer to control the % contribution of each pump is required. Analysis time decreases.

Generally the peaks are thinner and taller.

Question 12

Describe quantative and qualatative analysis in HPLC

Answer 12

Most HPLC detectors are used for quantitative analyses.

Some detectors may be used in a quantitative and qualitative (identity) mode.

The simplest qualitative analyses reports on the pattern and or number of HPLC peaks evident in the chromatogram (PROFILE).

Qualitative data also includes finding the identity of analyte(s) within each HPLC peak in a chromatogram. This is known as detector selectivity.

Question 13

What is detectory selectivity?

What are examples of detectors used?

In addition what can be used to increase detector selectivity?

Answer 13

The selectivity of a detector is its ability to determine an analyte of interest without interference from other materials present in the analytical system.

The more selective a detector, the lower the background noise recorded on the baseline.

• Refractive index detectors (RID) have low selectivity and defined as UNIVERSAL detectors
• UV and fluorescence detectors (UVD and FD) have much greater selectivity for their analytes
• Mass spectral detection (MSD) has probably the greatest selectivity of all.
• Chemical modification (derivatisation) of the analytes interest can be used to enhance detector selectivity.

Question 14

What is the sensitivity of a detector?

What does this include?

Answer 14

Sensitivity refers to the degree of response obtained from a detector.

Limit of detection (LOD) of a defined method is the smallest amount of analyte that is required for reliable determination.

• By definition LOD equates to a signal : noise ratio of 3:1. (The conc 3x the SD of the blank)
• Minium conc you can say “yes” this analyte is present

Limit of quantitation (LOQ) of a defined method is the smallest amount of analyte that can be reliably quantified.

• By definition LOQ equates to a signal : noise ratio of 10:1
• The conc 10x the conc of blank
• Minium conc you can accurately report

Question 15

Describe what is meant by linear and dynamic range of detector response

Answer 15

LINEAR RANGE = analyte signal is directly proportional to the amount of analyte present.

DYNAMIC RANGE = analyte signal is indirectly proportional to the amount of analyte present

Need to be in linear range to determine sensitivity

Question 16

Describe the use of a Refractive index detector

Answer 16

The reference cell is filled with mobile phase and the analyte passes through the sample cell, the refraction difference with time is plotted as chromatogram.

UNIVERSAL DETECTOR – will respond to anything (physical not chemical).

NOT ROBUST——e.g. sensitive to changes in temperature and the mobile phase composition. i.e. cannot be used for gradient runs.

SENSITIVITY LIMITED to over 1 µg

Question 18

What are chromophores?

What happens in them?

Answer 18

A chromophore is a region in a molecule where the energy difference between 2 different molecular orbitals falls within the ultraviolet or visible spectrum.

Specific and characteristic wavelengths of light that hit a chromophore can be absorbed by exciting an electron from its ground into an excited state.

In conjugated chromophores, the electrons jump between eight energy levels that are extended pi orbitals created by a series of alternating single and double bonds.

Others include C=O, N N, N-H, and N=O

Question 19

What UV detectors are available?

Answer 19

• FIXED wavelength e.g. set at 254 nm
  
  • Most organic compounds absorb here
  • Doesnt allow flexibility e.g. if compound not absorb here - use variable
• VARIABLE wavelengths - more modern, operates over full UV/Visible range
• Diode array detector

Question 20

What is the sensitivity of a UV/VIS detector?

Is it robust?

Answer 20

• ROBUST
• GOOD SENSITIVITY - (0.01 - 100 µg)

Question 22

How does a diode array detector differ and what can it do?

Answer 22

A sophisticated form of UV-Vis detector.

Operation:

A series of photodiode arrays enable the collection of the complete UV/Vis spectrum several times across the width of the HPLC peak

Can be used as a means of confirming peak identity and peak purity. E.g. is the spectrum the same at the front and tail of the separated peak.

Question 23

What are diode arrays useful for analysis off?

Answer 23

Useful for analyses:-

• where chromatographic peaks co-elute (but have different ʎ’s of absorbance)
• and PURITY of chromatographic peaks by WAVELENGTH RATIO ANALYSIS

Question 24

What is wavelength ratio analysis used for and how is it done?

Answer 24

Wavelength ratio analysis Enables an estimation of peak purity across an LC peak.

Here on-line spectra are collected at designated intervals across the HPLC peak.

As a minimum spectra are usually collected at the start of the HPLC peak, peak apex and peak tail.

Question 25

What detectors can be used if no chromophores

Answer 25

1. Use a refractive index detector - with resultant decrease in sensitivity
2. DERIVATISE (CHEMICALLY MODIFY) THE SAMPLE TO INTRODUCE A CHROMOPHORE.
   
   • May enhance sensitivity - up to 300X
   • May also enhance selectivity.
3. Add a fluorescent tag - increased sensitivity relative to many UV-Vis methods.

Question 26

Pre and post derivitization

Answer 26