Chromatography

Application and
Method development
Jeerapat Doungchawee, MsC (pharm).
Department of Food and Pharmaceutical Chemistry
Faculty of Pharmaceutical Sciences
Chulalongkorn University
27 February 2018
 Today’s Goal

Ability to explain and understandhow

to apply of chromatography in quantitative
and qualitative analysisthrough method d
evelopment and quality control
https://www.americanlaboratory.com/913-Technical-Articles/1569-Effective-UPLC-
Implementation/
IMPACT OF MIXING
Dwell volume (system volume) is measured from where the
mobile phase begins mixing to when it reaches the column
The larger of this volume, the longer it takes for the new
mobile phase conditions to arrive at the column
High Pressure Mixing Low Pressure Mixing
BINARY PUMPS QUATERNARY PUMPS
Mixing chamber lower Larger dwell volume lead to longer
dwell volume time for re-equilibration condition
• Deliver changes in the • Dwell volume take these volume
gradient much more into account
rapidly to the column • Volume from valves, pump
• Faster re-equilibration heads, pump head to column,
between samples additional injection, additional
mixer
 PROBLEM
The following chromatogram was obtained using a C18
column and a methanol: water as mobile phase.
How would the chromatogram change if
a. more ethanol were added to the sample
b. water was added to the sample
c. an unretained species was added to the sample
 IMPACT ON RETENTION TIME
Are the following changes in conditions likely to increase
or decrease retention time?
a. Faster flow rate
b. Longer column
c. Longer connector between column and detector
d. Higher analyte concentration
e. Higher temperature
f. Larger sample size
g. Using a less polar column while doing a reverse-phase
separation
h. using a less polar mobile phase while doing a reverse-
phase separation
Fronting peak

Column overload
• Fronting peaks are often caused by sample loads which
either exceed the buffer capacity of the mobile phase
or which exceed the solubility of the analyte.
The excess sample tends to stick to the stationary phase.
The result is that the top (high concentration) part of the
peak moves more slowly than the tailing (low
concentration) edge of the peak.

Dilute the sample by a factor of 10 (adjust detector range

as necessary) and re-inject.
• If the peak shape improves and the retention time (for
the top of the peak) …………………., the overload is
confirmed.
If the analyte is ionizable (weak acid or base), check the
pH and buffer concentration of the mobile phase.
• Flat-top peaks are often caused by sample loads that
exceed the linear range of the detector.
• Tailing peaks : sample loads that exceed the
capacity of the column. As the stationary phase
surface becomes saturated with analyte
molecules, the excess sample tends to stay in the
mobile phase and elute more quickly. The result
is that the top (high concentration) part of the
peak moves faster than the leading edge (low
concentration) of the peak.
• Dilute the sample by a factor of 10 (adjust
detector range as necessary) and re-inject. If the
peak shape improves and the retention time (for
the top of the peak) increases, the overload is
confirmed.
• METHOD DEVELOPMENT
• METHOD VALIDATION
• ADJUSTMENT
QUESTION TO CONSIDER BEFORE YOU
START METHOD DEVELOPMENT
What is the primary objective ?
• Qualitative analysis
• Quantitative analysis
• Detection of undesired
substances/ Purity determination
• Development of a new method
etc.
QUESTION TO CONSIDER BEFORE YOU
START METHOD DEVELOPMENT

What are the challenges ?

• Which component must be resolved ?
• Is sensitivity an issue
• Interferences ?
• What level of accuracy and precision
are required ?
• Is analysis time a significant factor?
etc.
 MATRIX
SAMPLE
• Bulk properties
• Complexity
• Processing consideration

ANALYTES
• Number of compounds targeted for
characterization
• Stability, volatility, solubility and molecular
weight
• Neutral or ionic
• Concentration rage
 General
Method selection consideration
• LC or GC
• Detection?
• MS vs MS/MS SAMPLE PROCESSING
BASIS FOR QUANTITATION
Miscellaneous considerations
• Sample quantity and availability
• Availability of reference standards and
internal standards
• Stability
• MW CHOICE OF HPLC
• Water
solubility
• Polarity
• Ionic or
non-ionic
 FIRST STEP
The best way to start is to carry out a
thorough literature search existing method

Compare existing method

Will an existing method solve your measurement need ?
Is the method practical or robust ? Validated?
Are there problems with the method ?
What are the reported specifications
Is the method applicable to the target sample matrix?
 Evaluate potential method (s)
• Prepare a solution containing the constituents
of interest
• Assess resolution and peak shape for target
analytes
• Prepare a sample from a typical sample matrix
• Assess interference
Consider improvement to method
• New technologies
• Modification of conditions
• Use simple solutions of analytes initially, then
more complex matrix based sample
Evaluate method performance
 METHOD VALIDATION
PARAMETERS FOR VALIDATION OF METHODS
• Linearity
Method’s Ability to obtain test result which
are proportional to concentration of analyte in the
sample
• Range
Range is the interval between the high and the low
Level of analyte studied
 Calibration procedure
The relation between the evaluated signal (y-
axis) and the amount of substances
(concentration, mass etc.) (x-axis) is determined
and calibration function is calculated.
 METHOD VALIDATION
PARAMETERS FOR VALIDATION OF METHODS
• Specificity / Selectivity
The analyte should have no interference from
other extraneous component and be well
resolved from tem.
For the drug substance or raw material, the related substances
to consider are process impurities (which include isomeric
impurities) from the synthesis process, residual pesticides,
solvents, and other extraneous components from extracts of
natural origin
e.g., 0.1% degradation products, the parent peak should be of a
size that at least a 0.1% detectability or area count is feasible.
 METHOD VALIDATION
PARAMETERS FOR VALIDATION OF METHODS

• Accuracy
Accuracy is the measurement of how close the
experimental value is to the true value
Recovery
Recovery is expressed as the amount/weight
of the compound of interest analyzed as a
percentage to the theoretical a mount
present in the medium.
 METHOD VALIDATION
PARAMETERS FOR VALIDATION OF METHODS
• Precision
is the measurement of how close the data
values are to each other for a number of
measurements under the same analytical
conditions
Repeatability Reproducibility Intermediate
Between laboratories precision
as in collaborative Different environment
studies depending on time and
resources
 METHOD VALIDATION
PARAMETERS FOR VALIDATION OF METHODS
• Detection limit and Quantitation limit
Detection limit (LOD) Quantitation limit (LOQ)
The lowest concentration The lowest concentration
of analyte in a sample that of analyte in a sample that
can be detected, but not can be determined with
necessarily quantitated, acceptable precision and
under the stated accuracy under the stated
experimental conditions. experimental conditions.
Chromatographic : Signal to noise ration = 2 or 3 (LOD), 10 (LOQ)
System suitability Specifications and Tests
System suitability tests are an integral part of GC and LC
methods. These tests are used to verify that the
chromatographic system is adequate for the intended
analysis.
The tests are based on the concept that the equipment,
electronics, analytical operations, and samples analyzed
constitute an integral system that can be evaluated as such.
Factors that may affect chromatographic behavior include the following:
• Composition, ionic strength, temperature, and apparent pH of the mobile phase
• Flow rate, column dimensions, column temperature, and pressure
• Stationary phase characteristics, including type of chromatographic support
(particle-based or monolithic), particle or macropore size, porosity, and specific
surface area
• Reverse-phase and other surface modification of the stationary phases, the
extent of chemical modification (as expressed by end-capping, carbon loading,
and others)
(General chapter <621>, USP40)
 System suitability parameter

Relative retention : location of two peaks.

Not an essential parameter as long as the resolution is stated

The ratio of the adjusted retention time of a component

relative to that of another used as a reference, obtained
under identical conditions: r = (tR2 − tM)/(tR1 − tM)

Relative retention time (RRT):

Also known as the “unadjusted relative retention”.
Comparisons in USP–NF are normally made in terms of
unadjusted relative retention, unless otherwise indicated.

RRT = tR2/tR1

(General chapter <621>, USP40)

Rret = b/c
 System suitability parameter

Precision /Injection repeatability -> %RSD

indicates the performance of the HPLC chromatograph
which includes the plumbing, column, and environmental
conditions, at the time the samples are analyzed.

Unless otherwise specified in the individual monograph,

• data from 5 replicate injections of the analyte are
used to calculate the relative standard deviation
(RSD), if the requirement is <2.0%
• data from 6 replicate injections are used if the RSD
requirement is >2.0%.

(General chapter <621>, USP40)

 Precision -> %RSD
For the assay in a drug substance monograph, where the value is
100% for the pure substance, and no maximum RSD is
stated, the maximum permitted %RSD is calculated for a series of
injections of the reference solution:

K is a constant (0.349), obtained from the

expression K = (0.6/ 2) x (t90%,5/ 6), in which 0.6/ 2 represents
the required %RSD after 6 injections for B = 1.0;
B is the upper limit given in the definition of the individual
monograph − 100%;
n is the number of replicate injections of the reference solution (3
≤ n ≤ 6);
t90%,n−1 is the Student’s t at the 90% probability level (double
sided) with n−1 degrees of freedom.
(General chapter <621>, USP40)
Unless otherwise prescribed, the maximum permitted
RSD does not exceed the appropriate value given in
Table 1 of repeatability requirements. This requirement
does not apply to tests for related substances.

RSD Requirement

Number of individual injections

3 4 5 6
B(%) Maximum Permitted RSD
2.0 0.41 0.59 0.73 0.85
2.5 0.52 0.74 0.92 1.06
3.0 0.62 0.89 1.10 1.27
 System suitability parameter
Theoretical plate number: Measure column efficiency ,
how many peaks can be located per unit run of chromatogram
For Gaussian peaks, it is calculated by:
N = 16(tR/W)2
Where electronic integrators are used, it may be convenient to
determine the number of theoretical plates, by the equation:

For a given stationary phase and mobile phase, N may be specified

• to ensure that closely eluting compounds are resolved from each
other, to establish the general resolving power of the system,
• to ensure that internal standards are resolved from the drug.
• Column efficiency is, in part, a reflection of peak sharpness,
which is important for the detection of trace components.

(General chapter <621>, USP40)

Resolution RS = 2 x (tR2 − tR1)/(W1 + W2)
Where electronic integrators are used, it may be
convenient to determine the resolution, by the equation:
RS = 1.18 x (tR2 − tR1)/(W1,h/2 + W2,h/2)
Retention factor (k):1 Also known as the “capacity factor
(k’)”. Defined as:

k’= (tR − tM)/tM

Retention volume (VR): The volume of mobile phase
required for elution of a component. It may be calculated
from the retention time and the flow rate in mL/min:
VR = tR x F

(General chapter <621>, USP40)

 Symmetry factor (As) or Tailing factor:
AS = W0.05/2f

As peak symmetry
moves away from
values of 1,
integration, and
hence precision,
become less reliable.

where W0.05 is the width of the peak at 5% height and f is the

distance from the peak maximum to the leading edge of the
peak, the distance being measured at a point 5% of the peak
height from the baseline
(General chapter <621>, USP40)
Example of type and cause Peak tailing
 Peak-to-valley ratio (p/v):
p/v may be employed as a system
suitability criterion in a test for
related substances when
baseline separation between two
peaks is not achieved.
This figure represents a partial
separation of two substances, where
Hp is the height above the
extrapolated baseline of the minor
peak
Hv is the height above the
extrapolated baseline at the lowest
point of the curve separating the
minor and major peaks:
p/v = Hp/Hv
(General chapter <621>, USP40)
 The signal-to-noise (S/N).

S/N ratio = 2H/h

H is the height of the peak measured from the peak apex to a

baseline extrapolated over a distance ≥ 5 times the peak width at
its half-height;
h is the difference between the largest and smallest noise values
observed over a distance ≥ 5 times
the width at the half-height of the peak and, if possible, situated
equally around the peak of interest
(General chapter <621>, USP40)
 Method adjustments
These system suitability tests are performed by collecting
data from replicate injections of standard or other solutions
as specified in the individual monograph.

Adjustments to the specified

chromatographic system may be necessary
in order to meet system suitability
requirements.
Adjustments to chromatographic systems performed in
order to comply with system suitability requirements
are not to be made in order to compensate for column
failure or system malfunction.

(General chapter <621>, USP40)

Adjustments of operating condition
If adjustments of operating conditions are necessary in order to
meet system suitability requirements, each of the items in
the following list is the maximum variation that can be
considered, unless otherwise directed in the monograph; these
changes may require additional verification data.

In some circumstances, it may be desirable to use an

HPLC column with different dimensions to those
prescribed in the official procedure (different length,
internal diameter, and/or particle size).
In either case, changes in the chemical characteristics
(“L” designation) of the stationary phase will be
considered a modification to the method and will require
full validation.
(General chapter <621>, USP40)
 Example list of adjustments of operating condition

Column length (GC): Can be adjusted as much as ± 70%.

Column inner diameter (GC): Can be adjusted by as much as
± 50%.
Film thickness (capillary GC): Can be adjusted by as much as
−50% to 100%.
Particle size (GC): Changing from a larger to a smaller or from
a smaller to a larger particle size GC mesh support is acceptable
if the chromatography meets the requirements of system
suitability and the same particle size range ratio is maintained.
The particle size range ratio is defined as the diameter of the
largest particle divided by the diameter of the smallest particle.
Flow rate (GC): The flow rate can be adjusted by as much as
± 50%. [NOTE—When the monograph specifies a linear
velocity parameter, the allowed velocity adjustment is between
+50% and −25%, provided the carrier gas system can be
maintained under control at the desired set points.]
 Example list of adjustments of operating condition

Particle size (HPLC)

For isocratic separations, the particle size and/or the
length of the column may be modified provided that
the ratio of the column length (L) to the particle size
(dp) remains constant or into the range between
−25% and 50% of the prescribed L/dp ratio.

Alternatively (as for the application of particle-size

adjustment to superficially porous particles),
other combinations of L and dp can be used provided
that the number of theoretical plates (N) is within
−25% to 50%, relative to the prescribed column.
 Example list of adjustments of operating condition

Column temperature (HPLC): The column temperature can be

adjusted by as much as ±10◦C. Column thermostating is
recommended to improve control and reproducibility of retention
time, which applies to both gradient and isocratic separations.
Oven temperature (GC): The oven temperature can be
adjusted by as much as ±10%.
Oven temperature program (GC): Adjustment of temperatures
is permitted as stated above. When the specified temperature
must be maintained or when the temperature must be changed
from one value to another, an adjustment of up to
± 20% is permitted.
Unless otherwise directed in the monograph, system suitability
parameters are determined from the analyte peak.

(General chapter <621>, USP40)

 Example list of adjustments of operating condition

Flow rate (HPLC): When the particle size

is changed, the flow rate may require
adjustment, because smaller-particle
columns will require higher linear
velocities for the same performance (as
measured by reduced plate height). Flow
rate changes for both a change in column
diameter and particle size can be made by:

F2 = F1 x[(dc22x dp1)/(dc12 x dp2)]

 F2 = F1 x[(dc22x dp1)/(dc12 x dp2)]

Original condition Modified conditions

Flow rates F1 F2 Changes in F,
dc, and dp are
Column diameters dc1 dc2 not allowed
for gradient
Particle size dp1 dp2 separations.

• When a change is made from≥ 3-µm to <3-µm particles in

isocratic separations, an additional increase in linear
velocity (by adjusting flow rate) may be justified, provided
that the column efficiency does not drop by >20%.
• Similarly, a change from <3-µm to ≥ 3-µm particles may
require additional reduction of linear velocity (flow rate) to
avoid reduction in column efficiency by>20%.
The flow rate can be adjusted by ± 50% (isocratic only).
Table from general chapter <621> USP40
Examples

A monograph specifies a 150-mm x 4.6-mm; 5-

µm column operated at 1.5 mL/min
Column we have is 75-mm x 2.1-mm; 2.5-µm
column, What flow rate should be used for the
same separation ?
F2 = F1 x[(dc22x dp1)/(dc12 x dp2)]
Or from Table
The same separation may be expected operated at
1.5 mL/min x 0.4 = 0.6 mL/min, along with a pressure
increase of about four times and a reduction in run
time to about 30% of the original.
Adapted from www.chromacademy.com

With the focus on packed columns,

reduction of Eddy Diffusion by using
-well packed columns
-smaller stationary phase particles
-particles with a narrow size
distribution
Adapted from www.chromacademy.com

Separation in HPLC with

packed column: Laminar flow
Adapted from www.chromacademy.com
Reduction of Axial Diffusion by using
-higher mobile phase flow rates
-short and narrow system tubing
(ideally <0.12mm i.d. but this also
causes higher back-pressure)
-particles with a narrow size
-correct nuts, ferrules and fittings

Very low flow or peak

parking for hours
Adapted from www.chromacademy.com

Reduction of mass transfer effect by

using
-smaller stationary phase particles
-lower mobile phase flow rates
-separation at higher temperatures
(faster diffusion)

Slow mass transfer

equilibrium or too high flow

Fast mass transfer

equilibrium or low flow
 Example list of adjustments of operating condition

pH of mobile phase (HPLC):

The pH of the aqueous buffer used in the preparation of
the mobile phase can be adjusted to within ± 0.2 units of
the value or range specified. Applies to both gradient and
isocratic separations.

Concentration of salts in buffer (HPLC):

The concentration of the salts used in the preparation of
the aqueous buffer employed in the mobile phase can be
adjusted to within ± 10% if the permitted pH variation is
met.
Inconsistent and tailing peaks may occur when operating
close to an analyte pka and should be avoided
QUALITATIVE ANALYSIS
What is Qualitative information

• Qualitative analysis involves running a

standard that contains the target analytes.
We note retention time
• Standard that are used for calibrating an
instrument are called “Calibration
standards” for obvious reason
• HPLC software can build a calibration table
containing retention times for the analytes
we are interested in.
 Example of Qualitative test
Run the sample(s) of interest. If we see peaks
present in the sample that correspond to peaks
in our calibration standard, we have a
probability of a match.

• For example, if we ran a calibration standard for

200 DOPING AGENTS and checked retention
times were a,b,c….x minutes and then ran a
URINE sample from Subject A and
saw a peak at X minutes, we might say that
Subject A had doped! we would want to confirm
that before we made any accusations.
 Example of Qualitative test

Mebendazole
 (USP40)

Standard solution = 10
mg/mL
Sample solution
200 mg/20 mL = 10
mg/mL
 Retardation factor (Rf) = Distance solute moved (b)
Distance solvent moved (a)
Solvent
front
Solvent front = 15.2 cm

a Item Distance Rf
(cm)
b Standard 10.41
Sample A 10.35
STD A B
Immersion Sample B 10.38
line

Conclusion :
 Example of Qualitative test

https://pubchem.ncbi.nlm.nih.gov

Cefotaxime
QUANTITATIVE ANALYSIS
What is Quantitative information
• Full quantitative analysis can be called
validation of chromatographic method
Guidelines
• Food and Drug Administration (FDA)
• International Conference on Harmonisation of
Technical Requirements for Registration of
Pharmaceuticals for Human Use (ICH)

Q3A(R) Impurities in New Drug Substances (Revised)

chemistry and safety aspects of impurities, listing of
impurities in specifications and defines the thresholds for
reporting, identification and qualification.
Quantification
Detector response. The relative detector
response factor, commonly referred to as
response factor, expresses the sensitivity
of a detector relative to a standard
substance

Draw chromatogram peak of double the concentration

A separation produced the chromatogram shown
below. Overtop of the chromatogram, sketch show
what it would look like if a second sample with more
(higher concentration) of analyte was run.
Example of Quantitative test
Example of Quantitative test
Example of Quantitative test
 Standards
• A reference standard is a highly purity
compound that is well characterized.
• Chromatographic methods rely heavily
on a reference standard to provide
accurate data.
• Therefore the quality and purity of the
reference standard is very important.
 External standard method
• An external standard method is used
when the standard is analyzed on a
separate chromatogram from the
sample.
• Quantitation is based on a comparison
of the peak area/peak height of the
sample to that of a reference standard
of the analyte of interest.
External standard method
Is more appropriate for sample as follows

• Sample with a single target

concentration and narrow concentration
range. e.g. acceptance and release test
• Simple sample preparation procedure
• Increased baseline time for detection of
potential extraneous peaks. e.g.
impurities test.
Example of Quantitative test
Example of Quantitative test
Example of Quantitative test
http://static.usp.org/pdf/EN/referenceStandards/certificates/1097909-R071G0.pdf
 Internal standard method
• With an internal standard method,
compound of known purity that does not
cause interference in the analysis is added
to the sample mixture.
• Quantitation is based on the response ratio
of compound of interest to the internal
standard and response ratio of a similar
preparation of the reference standard
methods to the internal standard
 Internal standard method
Is more appropriate if it can be implemented

• Complex sample preparation

procedures, e.g. multiple extractions
• Low concentration sample (Sensitvity
being an issue) e.g. pharmacokinetics
studies.
• Wide range of concentrations expected
in the sample for analysis. e.g.
pharmacokinetics studies.
 Standard Addition
• Must be used whenever the matrix of a sample
changes the analytical sensitivity of the method.
(the slope of the working curve for standards made
with distilled water is different from the same
working curve made up in matrix happens to be).
Standard addition vs Internal standards
Raw material Finished product/
Bulk product
The result is The result is
calculated on the generally
• As is basis calculated as
• Dried basis % Labeled
• Anhydrous basis amount
 Finished
Assay product/ Bulk
e.g. Contains Product
paracetamol
99.9% of the
labeled Dissolution
amount 85.0% of the
Content uniformity
(499.5 mg) Use content (%L.A.) of labeled amount
of Paracetamol
each unit to determine is dissolved in
the uniformity of each 30 minutes
batch
 Finished product/
Bulk Product

LA = 10 mg/capsule

USP40

What would be the permissible range in content of nifedipine,

expressed in mg?
To Calculate % Labeled amount
From experiment, test

Amount of drug (theoretical)

according to label amount

Amount of drug found

Which form to calculate ?
(Base, Salt, Anhydrous, Monohydrate etc.)
Assay or Dissolution or Content uniformity etc.
 Sample solution
Amount of drug found

Amount of drug in dosage unit

Assay Sample solution varied
Content uniformity Sample solution is usually use one unit

Amount of drug in dosage unit that is dissolved

Dissolution Sample solution :
1 tablet is dissolved in vessel in specific
condition

TEST 6 TABLETS
Ex. A Tablet à Each tablet contains Paracetamol 500 mg Assay
USP40

Calculate % Labeled amount of acetaminophen

Assay of Acetaminophen tablet Assay

Standard
solution
HPLC
(Instrument)
Monograph : 0.1 mg/ml
Response : rs

Sample
solution HPLC
(Instrument)
Monograph : 0.1 mg/ml
Responese : ru
 Information Assay
Acetaminophen reference standard
100.5% of C8H9NO2 , calculated on
the dried basis
%Loss on drying (%LOD) = 0.3%

2 mL
Accurately
weighed
25.50 mg

50-mL 100-mL Standard

solution
 Assay
Standard
solution

Calculate concentration of standard solution

= 0.010220 mg/mL
 Assay
2 mL

20 tablets

Weight of 20
tablets = 100-mL
10.9500 g 114.45 mg
(weight/tablet 200-mL
= 547.50 mg)

Sample
? g of powder equivalent to 100 solution
mg of acetaminophen?
 Assay
Result
Solution Weight Respons
(mg) e

Standard 25.50 0.010220 0.85

Sample 114.45 Cu = ? 0.86

Rearrangement from Beer’s law

ru = response from sample solution
Sample
rs = response from standard solution
solution Cs = concentration of standard (mg/mL)
Cu = concentration of sample(mg/mL)
 Assay
2 mL

20 tablets

Weight of 20
tablets = 100-mL
10.9500 g 114.45 mg
(weight/tablet 200-mL
= 547.50 mg)

Sample
? g of powder equivalent to 100 solution
mg of acetaminophen?
 Assay

%Labeled amount = X 100

Calculate concentration of sample solution Sample solution
à amount of drug found (mg)

2 mL

200-mL
100-mL

= 103.2 mg
Label amount : 1 tablet contains 500 mg of paracetamol
Weight of 20 tablets = 10.9500 g
Weight of 1 tablet = 547.50 mg

Powder 547.5 mg à 500 mg of acetaminophen

= 104.56 mg of acetaminophen
 Assay

%Labeled amount = X 100

= 98.69 % of the labeled amount

In summary, ru

rs Cs
1/Cu
(Monograph)
Calculate the %LA of C8H9NO2 in the portion of tablets
taken:
Result = (ru/rs) x (Cs/Cu) x 100
 Gas Chromatography
• Residual solvent
 Gas Chromatography
• Residual solvent
TIPS
 Improve Selectivity

pH Robustness (Slightly change of pH)

The resolution of ionizable compounds can change

markedly with pH changes—even as small as 0.05–
0.25 pH units.
 Improve Selectivity

Effect of pH on Peak shape

Inconsistent and tailing peaks may occur when operating

close to an analyte’s pKa and should be avoided.
 Improve Selectivity
Similar stationary phase may
give different selectivity
 Improve Chromatographic choices

• Shorten analysis time:

– reduce column length,
– increase flow rate
• Sample Preparation
 Improving Resolution

• Increasing k (retention factor)

– Change temperature (GC)
– Change MP composition (LC)
• Increasing N (number of plates)
– Lengthen column (GC)
– Decrease particle size of
stationary phase (LC)
• Increasing selectivity factor
– Changing mobile phase
– Changing column temperature
– Changing stationary phase
 Question to practice

1.Describe the chromatographic nature (there is a

particular term that describes each) of the problem.
2. Propose a way in gas chromatography to
eliminate both problems.
3.Propose a way in liquid chromatography to
eliminate both problems.
Where are you now?
Chromatography EXAM
Total 40 , 18.5%
• True-False : Chromatography(10)
• Fill-in (30)
– HPLC (10)
– GC (10)
– TLC (5)
– General and HPLC (5)