Python Notes for mca i year students osmania university.docx
hplcyunes-190424112733 (2).pdf
1. HPLC
(Peak shapes, capacity factor, selectivity,plate
number,plate height,resolution and band
Braodening )
Presented by_Yunes Alsayadi Presented to - Dr.Rahul Kumar
M.Pharm(Analysis)
2nd sem,ISF College of
Pharmacy, Moga, Punjab
2.
3. Introduction to Peak Shapes in HPLC
One of the aims of chromatographers everywhere is to get nice sharp Gaussian
peaks for each of the components in the run
The ideal is a Gaussian or symmetrical shaped peak, a narrow peak width at half-
height when compared to its height and no peak fronting, tailing or broadening.
Poor peak shape can cause integration and resolution errors in your analysis —
which ultimately means poor analysis and wasted time and money.
Have you ever encountered a situation during HPLC analysis when you thought:
"something is wrong with these peak shapes."?
Abnormal peak shapes are a common problem when conducting routine analysis
work. Peak abnormalities that are clearly noticeable in chromatograms include
peak broadening (including extreme tailing or leading edges), shoulder peaks, and
split peaks
4. Peak Asymmetry
Asymmetrical peaks are more difficult to
resolve, therefore, integration of the peak to
provide a peak area for quantitation will also
be much less reproducible.
Tailing describes a peak whose tail portion
(distance ‘B’ in the diagram) is wider than
the front portion (distance ‘A’ in the
diagram).
Due to the effects of instrument dead-
volume, adsorptive effects of the stationary
phase and the quality of the column packing,
peaks may often show a tailing behavior.
Also, if the sample concentration is too high
or if the column is damaged and contains
‘channels’ then a fronting peak shape may
occur.
5. What is Good Peak Shape and Why is it
Important ?
• Good peak shape can be defined as a symmetrical or gaussian peak
and poor peak shape can include both peak fronting and tailing.
Good peak shape can be defined by….
Tailing factor of 1.0
High efficiency
Narrow peak width
Good peak shape is important for….
Improved resolution (Rs)
More accurate quantitation
Longer usable column lifetime (based on system suitability criteria)
6. How is Peak Shape Measured?
• • Measures
• • USP Tailing Factor – at 5% of peak
height*
• • Asymmetry – at 10% of peak height
• • Indicators
• • Efficiency – plates*
• • Peak Width – peak width at ½
height*
7. Factors Affecting Peak Shape
Column packing factors
• Silica type
• Bonded phase and endcapping
Mobile phase factors
Sample factors
8. High Purity, Low Acidity Silica
Improves Peak Shape
Standard Silica
High Purity, Low Acidity ZORBAX
Rx-SIL
9. Mobile Phase Factors for Improved
Peak Shape
pH
Inconsistent and tailing peaks may occur when operating close to an analyte pKa and
should be avoided.
Buffers
Buffered mobile phases enhance retention, resolution, and peak shape.
Organic modifiers
Changing the organic modifier may improve peak shape due to secondary interactions.
Additional mobile phase(using modifiers (TEA, TFA)
14. Ghost Peaks
Ghost peaks are contaminant peaks that appear
even when no sample is injected. There are many
causes for ghost peaks and this note will describe
how to troubleshoot these contaminant peaks,
when you see them.
The primary cause of a ghost peak is a dirty pre-
column or column. Remove the pre-column and
run a sample. If the ghost peaks are no longer
present, replace the pre-column (or frit).
15. Sample and Additional Considerations for
Good Peak Shape
Injecting in a solvent stronger than the mobile phase can
cause peak shape problems, such as peak splitting or
broadening.
Sample Overload May Cause Peak Fronting and Tailing
Dead Volume in Flow Lines
Dead volume is the total volume of the liquid phase in
the chromatographic column.
16. Guidelines for Improved Peak Shape
Select columns based on high purity fully hydroxylated silica –
Zorbax Rx-Sil based columns.
Select double endcapped columns for mid pH or difficult basic
compounds, such as Eclipse XDB.
Select wide-pore columns for high molecular weight analytes
Use buffered low pH mobile phases to reduce secondary
interactions
Use additional additives only when needed
Check sample solvents
17. Retention (Capacity) Factor (k)
The retention (or capacity) factor (k) is a means of measuring the retention of an
analyte on the chromatographic column.
• Retention time (RT) is the difference in time between the point of injection and
appearance of peak maxima.
• It is also defined as time required for 50% of a component to be eluted from a
column.
• It is measured in minutes and seconds.
• The retention time is longer when the solute has higher affinity to the stationary
phase due to its chemical nature.
18. A high k value indicates that the
sample is highly retained and has spent
a significant amount of time interacting
with the stationary phase.
The retention factor is equal to the
ratio of retention time of the analyte
on the column to the retention time of
a non-retained compound.
The non-retained compound has no
affinity for the stationary phase and
elutes with the solvent front at a time
t0, which is also known as the ‘hold-up
time’ or ‘dead time.
19. How to change Retention (Capacity) Factor (k)
The most effective and convenient way to alter the retention factor of a peak is to
adjust the ‘solvent strength’ of the mobile phase.
Characteristically reversed phase HPLC has a non-polar stationary phase,
therefore, increasing the polarity of the mobile phase will increasingly repel the
hydrophobic (nonpolar) sections of the analyte molecules into the stationary
phase and the analyte will be retained for longer on the column.
The largest gain in resolution is achieved when the k value is between 1 and 5.
k values less than 1 are unreliable as analytes may be eluting with other sample
components or solvent.
Too much retention wastes valuable analysis time and the chromatographic peak
height will decrease as the bandwidth of the peaks increases.
20. SELECTIVITY(SEPARATION) FACTOR (α)
The selectivity (or separation) factor
(α) is the ability of the
chromatographic system to
‘chemically’ distinguish between
sample components.
It is usually measured as a ratio of
the retention (capacity) factors (k) of
the two peaks in question and can be
visualized as the distance between
the apices of the two peaks.
By definition, the selectivity is always
greater than one – as when α is equal
to one, the two peaks are co-eluting
(i.e. their retention factor values are
identical).
21. High α values indicate good separating power and a good separation between the
APEX of each peak. However, the alpha value is NOT directly indicative of the
resolution.
Selectivity values Separation
≥ 2 Easy separation
1.5-2 Possible separation
1.2-1.5 Difficult separation
≤ 1.2 Very difficult separation
22. Effects of Selectivity on Resolution
Changing selectivity can have a dramatic effect on the chromatographic resolution
Selectivity is relatively simple to alter, with mobile phase constituents (solvent
type, ion pair reagents etc.) and pH being the most frequently used methods of
adjustment.
If suitable resolution cannot be achieved by altering the mobile phase
constituents, an alternative column chemistry should be investigated as a means
of altering the selectivity of the separation.
23. Theoretical Plate Number
The plate number (N) is a measure of the peak dispersion on the HPLC column, which
reflects the column performance.
Efficiency is derived from an analogy of Martyn and Synge who likened column
efficiency to fractional distillation, where the column is divided into Theoretical
PlatesThe efficiency of a column is reported as the number of theoretical plates (plate
number).
Each plate is the distance over which the sample components achieve one
equilibration between the stationary and mobile phase in the column.
where,
tr is the retention time measured from the instant of injection
w is the peak width
24. Poor chromatograms are those with early peaks (small tr) that are broad (large w),
hence giving small N values, while excellent chromatograms are those with late-
appearing peaks (large tr) that are still very narrow (small w), thereby producing a
large N.
The number of theoretical plates is a measure of the “goodness” of the column.
Plate numbers may range from 100 to 106.
The plate number depends on the length of the column.
A typical plate number for a 4.6 × 100 mm column with 5 μm particles is between
5000 and 8000.
A more appropriate parameter for measuring efficiency is the height equivalent to
a theoretical plate (or plate height)
25. Factors affecting column efficiency (plate
number)
Column length
Particle size
Packing quality
Linear velocity (flow)
Instrument quality (dead volume)
Retention factor
For a given column length, the plate number (Nth) is inversely related to the
particle size of the column packing. The smaller the particles, the higher the plate
number and the separation power.
The plate number is also dependent on the flow rate (F) of the mobile phase.
26. HEIGHT EQUIVALENT OF A THEORETICAL PLATE
(HETP)
A theoretical plate is an imaginary or hypothetical unit of a column where
distribution of solute between stationary phase and mobile phase has attained
equilibrium. It can also be called as a functional unit of the column.
The height of one theoretical plate is referred to as the ‘Height Equivalent of a
Theoretical Plate.
A theoretical plate can be of any height, which describes the efficiency of
separation. If HETP is less, the column is more efficient. If HETP is more, the
column is less efficient.
HETP = length of the column/ no. of theoretical plates
HETP is given by Van Deemeter equation
Best known is the van Deemter equation, which describes the various
contributions to plate height (H). In this equation the parameters that influence
the overall peak width are expressed in three terms:
27. where,
H = HETP (plate height)
A = eddy diffusion term
B = longitudinal diffusion term
u = linear velocity
C = Resistance to mass transfer coefficient
28. Peak height and peak broadening are governed by kinetic processes in the column
such as molecular dispersion, diffusion and slow mass transfer.
A-term: eddy diffusion: The column packing consists of particles with flow
channels in between. Due to the difference in packing and particle shape, the
speed of the mobile phase in the various flow channels differs and analyte
molecules travel along different flow paths through the channnels.
B-term: longitudinal diffusion: Molecules traverse the column under influence
of the flowing mobile phase. Due to molecular diffusion, slight dispersions of the
mean flow rate will be the result.
C-term: resistance against mass transfer : A chromatographic system is in
dynamic equilibrium. As the mobile phase is moving continuously, the system has
to restore this equilibrium continuously. Since it takes some time to restore
equilibrium (resistance to mass transfer), the concentration profiles of sample
components between mobile and stationary phase are always slightly shifted.
29. Resolution
The most important thing in HPLC is to obtain the optimum resolution in the minimum
time.
The resolution of a elution is a quantitative measure of how well two elution peaks can
be differentiated in a chromatographic separation.
It is defined as the difference in retention times between the two peaks, divided by
the combined widths of the elution peaks.
Where B is the species with the longer retention time, and tR and W are the retention
time and elution peak width respectively.
If the resolution is greater than one, the peaks can usually be differentiated
successfully.
30. A resolution value of 1.5 or greater between two peaks will ensure that the sample
components are well ('baseline') separated - to a degree at which the area or height
of each peak may be accurately measured.
• Resolution is calculated using the separation of two peaks in terms of their average
peak width at the base (t R2 > t R1).
31. Calculate the resolution, R, between two peaks by
R = (RT1 - RT2) / [0.5 * (W1 + W2)],
where RT1 and RT2 represent the retention times of peaks 1 and 2, and
W1 and W2 represent the widths of the peaks taken at their bases.
Example, if one peak exhibits a retention time of 16.8 seconds and a width
of 3.4 seconds. If the second peak exhibited a retention time of 21.4
seconds with a width of 3.6 seconds, then the resolution would be
R = (21.4 - 16.8) / [0.5 * (3.4 + 3.6)] = 4.6 / 3.5 = 1.3.
32. Band Broadening
• Band broadening is a phenomenon that reduces the efficiency of the separation
being carried out –leading to poor resolution and chromatographic performance
• The degree of band broadening (loss of efficiency) naturally increases with the age
of the chromatographic column being used
• broadening in chromatographic systems can be divided into two broad areas of
concern
• One is the contribution from what is known as dead volume and The other
source of broadening is within the column .
• Dead volume refers to all the volume in a chromatographic system from the
injector to the detector other than the column.
33. Broad peaks:
Possible cause:
Overloading of column
Retention time too long
Extra-column volume too large
"Dead" volume
Detector time too slow
Possible solution:
Dilute sample 1:10 and rerun
Modify gradient so that peaks
elute earlier
Reduce tubing diameters and
lengths where possible,
particularly post-column
Check all connections for proper
fit
Increase detector Hz rate
34. Peak Tailing:
Peak Fronting:
Possible cause: Possible solution:
Dead volume in flow
path
Check all fittings,
especially post-
column and at the
column head
Mobile phase too
weak
Remake mobile
phase or increase
acid concentration
Overloading of
column
Dilute sample 1:10
and rerun or use a
larger capicity
column
Column is poorly
packed
Run high organic
through column,
then equilibrate and
retry, or replace
column
