Study Guide
Column Chromatography and HPLC
-
University:
Kentucky State University -
Course:
CHE 303 | Quantitative Analysis Academic year:
2019
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Views:
300
Pages:
45
Author:
Quintin Ware
Introduction to Chromatography
Column chromatography and HPLC Basic of Chromatography
Chromatography is a separation technique to seprate mixtures into their
components in order to identify (qualitative analysis), purify and/or quantify
(quantitive analysis) the mixture or components.
Chromatography is the most frequently used analytical technique in
pharmaceutical analysis.
Chromatography aims to separate a mixture of chemical substances into its
individual components, so that the individual components can be thoroughly
analyzed.
Two Phases:
Stationary Phase (stays in a place): Liq or solid
Mobile Phase (flows through column or move in a plate, carries analyte): Liq or gas History of Chromatography
The Russian botanist Mikhail Tswett coined the term
chromatography in 1906 to describe his experiments in
separating different colored constituents of leaves through a
column.
”Chromatography”; Khroma (colour) and graphic (writing) Classification of Chromatography
Based on:
Mechanism of Separation: Normal or Reversed,
chromatography, affinity chromatography
ion-exchange
Shape of the stationary Phase: Column or Planer
The Physical State of the mobile phase: Liquid chromatography (LC) or
Gas chromatography (GC)
The purpose of separation: Analytical or Preparative • Planar chromatography: flat stationary phase, mobile phase
moves through capillary action or gravity.
- Thin Layer Chromatography (TLC)
- Paper Chromatography
• Column chromatography: tube of stationary
phase, mobile phase moves by pressure or
gravity Term
Definition
Mobile phase or Carrier
Solvent moving through the column
Stationary phase or Adsorbent Substance that stays fixed inside the column
Elution
The process of washing out a compound
through a column using a suitable solvent
Retention
The time the analyte spends interacting with
the stationary phase
Chromatogram
The visual output of chromatography
Chromatograph
Instrument used in chromatography
Analyte
Sample to be analyzed Basic of Chromatography
• There are two principal mechanisms which produce retardation of a compound passing
through a column:
1) A straight-phase packing, where the mechanism of retardation is by adsorption of the polar
groups of a molecule onto the polar groups of the stationary phase (Normal Phase).
2) ODS-coated silica gel, which is a reversed-phase packing, where the mechanism of
retardation is due to partitioning of the molecule into the stationary phase according to its
lipophilicity.
• The extent to which a compound is retained will depend primarily upon its polarity, in the
case of silica gel, and primarily upon its lipophilicity in the case of a reversed-phase packing
such as ODS silica gel. Normal Phase Vs Reversed Phase Chromatography
“Normal” phase LC:
• Polar stationary phase (Silica gel is the most commonly used)
• Non-polar mobile phase (Diethyl ether, dichloromathane)
• The extent to which a compound is retained will depend primarily upon its polarity. More
polar drugs will interact more with the stationary phase.
• Order of elution: non-polar polar
• Solvent (eluent) strength: non-polar (weak), polar (strong) The more polar a mobile
phase, the more quickly it will elute a compound from a silica gel column. Normal Phase Vs Reversed Phase Chromatography
“Reversed” phase LC:
• Non-polar stationary phase (ODS silica gel or C18)
• Polar mobile phase (Water, MeOH, ACN, THF)
• The extent to which a compound is retained will depend primarily upon its lipophilicity
• Order of elution: polar non-polar
• Solvent (eluent) strength: non-polar (strong), polar (weak) the more lipophilic a mobile
phase, the more quickly it will elute a compound from a reversed-phase column. Common stationary phases:
• Polar phases:
• Amino [R = (CH2)3NH2 ]
• Cyano [R = (CH2)3CN ]
• Diol [R = (CH2)2OCH2CH(OH)CH2OH ]
• Non-Polar phases:
• Octadecyl, C18 [R = (CH2)17CH3 ]
• Octyl [R = (CH2)7CH3 ]
• Phenyl [R = (CH2)3C6H5 ] Stationary phase Most drug molecules have both lipophilic and polar groups.
Interaction of naproxen with the surfaces of silica gel and octadecylsilyl
(ODS) silica gel HPLC packings Liquid Chromatography
Like dissolves Like!
• “Normal” phase LC:
• Polar stationary phase
• Non-polar mobile phase (Diethyl ether, dichloromathane)
• Order of elution: non-polar polar
• Solvent (eluent) strength: non-polar (weak), polar (strong).
• “Reversed” phase LC:
• Non-polar stationary phase
• Polar mobile phase (Water, MeOH, ACN, THF)
• Order of elution: polar non-polar
• Solvent (eluent) strength: non-polar (strong), polar (weak) .
The greater the eluent strength, , the more rapidly solutes
will be eluted Liquid Chromatography (LC)
• Column Chromatography
- Classical column chromatography
- High-performance liquid chromatography (HPLC): Column chromatography (CC)
• Alumina and silica are the two most popular stationary phases in CC. The more polar sample
will be retained on the stationary phase longer.
• The least polar compound will elute from the column first, followed by each compound in
order of increasing polarity.
• The type of adsorbent, the size of the column, the polarity of the mobile phase as well as the
rate of elution all affect the separation.
• Column chromatography can be used on both large and small scale. Columns can be as thin as
a pencil to a diameter of several feet in industrial processes. They can separate milligram to
kilogram quantities of materials.
• If we needed to collect the separated materials, CC could be used. Column chromatography
allows us to separate and collect the compounds individually (purification) • Isocratic system:
Means using the same mobile phase from the beginning to the end of the separation.
• Gradient system:
The polarity of the system increased gradually during separation by increasing the proportion of the
more polar solvent.
• Monitoring the column (mixture to be separated contains colored or colorless compounds):
The colored bands will move down the column along with the solvent and as they approach the end of
the column, collect the colors in individual containers. Use the color as your guide.
Usually fractions of certain volume are collected evaporated to small volume and spotted on TLC.
Similar fractions are collected together for more purification or crystallization. Liquid Chromatography: HPLC
• High-performance liquid chromatography (HPLC) is the
technique most commonly used for the quantitation of drugs in
formulations.
Classical:
HPLC:
Packing
50 m
3-5 m
Pressure
gravity
3000psi
N
500
15,000
• HPLC: higher resolution, faster and good quantification, but expensive and complex. HPLC: High Performance liquid Chromatography
1) A solvent reservoir (Mobile Phase)
2) A pump capable of pumping the mobile phase (pressure, flow rate)
3) Injector
4) A column, which is usually a stainless steel tube packed, usually, with
octadecylsilane-coated (ODS-coated) silica gel with an average particle diameter (3,
5 or 10 µm)
5) Detector, which is usually a UV/visible detector, although for specialist
applications a wide range of detectors is available.
6) A data capture system, which may be a computing integrator or a PC with
software suitable for processing chromatographic data. Instrumentation
• Reversed Phase (mobile
phase polar)
Solvent A: Water or Buffer
Solvent B: MeOH
ACN
THF HPLC columns Column Characteristics
1)
2)
3)
4)
5)
Stationary phase type (ODS, C4, C8)
Pore size: small pore silica (~ 100 angstrom), wide poresilica (~ 300 angstrom).
Particle size
Internal diameter: Narrow ( 2.1mm) - higher detector sensitivity, Wide (10-22mm) - high
sample loading
Length: Short (30-50mm): short run times, low backpressure, Long (250- 300mm) - higher
resolution, long run times Common stationary phases:
• Polar phases:
• Amino [R = (CH2)3NH2 ]
• Cyano [R = (CH2)3CN ]
• Diol [R = (CH2)2OCH2CH(OH)CH2OH ]
• Non-Polar phases:
• Octadecyl, C18 [R = (CH2)17CH3 ]
• Octyl [R = (CH2)7CH3 ]
• Phenyl [R = (CH2)3C6H5 ] Chromatogram
Detector
Response
tr2
tr1
tm
injection time
tr1
Time (min)
t0 or tm= time for mobile phase to travel length of column (dead time).
tr = retention time
tr = adjusted retention time = tr - tm • Isocratic system:
Means using the same mobile phase from the beginning till the end of the
separation (50:50, solvent A:solvent B)
• Gradient system:
• The composition of the mobile phase components is changing through the
chromatographic run
e.g. The polarity of the system increased gradually during separation by increasing
the proportion of the more polar solvent • Isocratic and Gradient System:
Benzene °=0.32
Acetonitrile °=0.65
Start with 100% Benzene
then add Acetonitrile
“isocratic
elution”
“gradient
elution” Peptide map of bovine serum albumin Chromatographic Parameters
Capacity factor (k)
k = (tr-tm)/tm
- ↑k = greater retention
Bigger K’ means that the material retained more time on the
column (move slowly). Smaller K’ means faster movement.
Optimum k value??
Relative retention (separation factor)
α = k2/k1
α > 1.
- indicates quality of the separation
- ↑α = greater separation. Column Efficiency
• It was assumed that the chromatographic system composed of number of
“distribution systems” or “equilibrations” called “Theoretical Plates”. Each
theoretical plat is composed of stationary phase and mobile phase.
• The number of theoretical plates “N” is important for separation. Increasing “N”
resulted in narrower bands and better separation.
N: Column efficiency
L: Column length
H: Height of theoretical plate
• “N” can be increased by:
1- Increase the length of the column.
2- Decrease the HETP.
- Decrease the particle size of the stationary phase.
- Proper selection of good mobile phase. • Example: The effect of increasing the % of organic solvent on the elution of a series of alkyl
benzenes in ODC. In methanol/water mixture Dolan’s rule of 3 applies where, in reversed phase
chromatography, a 10% decrease in methanol content produces a 3 times increase in capacity factor
for an analyte. Resolution of Separation
• We Want Resolution > 1.5
• The separation is worse with the increasing peak width • Example: the effect of increasing the % of organic solvent on the elution of a series of alkyl benzenes in ODS. 1) Long eluting peaks
2) Bad resolution between D and E Peak Tailing and Peak Fronting Factors affecting separation
• Factors related to stationary phase
1) Particle size of the stationary phase: Reducing the particle size increases the surface area and
improve separation.
2) Adsorbent activity: The choice of the suitable adsorbent is very important.
3) Uniformity of packing of the column: If the stationary phase is not packed uniformly then the bands
will be irregular and less uniform resulting in poor separation.
4) Concentration of the mixture: the proper ratio between sample to be separated and the amount of
stationary phase is very important too much samples resulted in bad separation. Factors related to Mobile Phase:
1) Selection of the proper mobile phase: Very polar mobile phase will wash out all components without
any separation. On the other hand very non polar mobile phase will result in broad band and poor
separation.
2) Flow rate: Slower flow rate usually resulted in a better separation and narrower bands.
Factors related to Columns:
1) Column dimensions: Increasing the length of the column improve separation. However, increasing
the column length some times is impractical.
2) Column temperature: Increasing the temperature usually reduces the adsorption power of the
stationary phase and increase elution speed. This may leads to decrease in the efficiency separation. Applications of HPLC
Qualitative Analysis (Identify analytes in your sample)
Retention time can be used to identify analytes
if the method conditions are constant. Quantitative analysis
• The area under a peak is proportional to the amount of analyte present in the
chromatogram.
• Area of the peak can be used to determine the concentration
of an analyte in the original sample.
• A calibration curve created by finding the
response for a series of concentrations of analyte, or by
determining the relative response factor (RRF)
of an analyte. Assay: Analysis of paracetamol tablets
Column Chromatography and HPLC
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