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HPLC
Tujuan Perkuliahan
Memahami prinsip pemisahan menggunakan
HPLC
Mengetahui instrument-instrument dalam
HPLC
Memahami prinsip pemilihan metode dalam
HPLC
Mampu merancang suatu penelitian
menggunakan instrument HPLC
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Detector
Column
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4
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Detector
Column
10
5
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11
12
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Pump head
Motor and cam
Check
valves
Plunger
Plunger seal 10 -100L
13
Check valves
Plunger head
14
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Plunger heads
Type Type
15
Gradient System
Isocratic system
Constant eluent composition
Gradient system
Varying eluent composition
HPGE (High Pressure Gradient)
LPGE (Low Pressure Gradient)
16
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In isocratic mode
CH3OH / H2O = 6 / 4
Long analysis time!!
95%
Concentration of methanol in eluent
30%
18
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Low-pressure
gradient unit
Mixer
Mixer
20
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Degasser
21
Online Degasser
To pump
To pump
To draft
Drain valve
22
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23
Manual Injector
From pump
To column
LOAD position
From pump
To column
INJECT position
24
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Manual Injector:
Operating Principle of Sample Injection
Loop
To column
To column
LOAD INJECT
25
Manual Injector:
Injection Method
Syringe measurement method
It is desirable that no more than half the loop
volume is injected.
Loop measurement method
It is desirable that at least 3 times the loop volume
is injected.
26
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Autosampler
(Pressure Injection Method)
Sample Loop
LOAD 27
INJECT
Autosampler
(Total-Volume Injection Method)
From pump To column From pump To column
Needle
Sample vial
LOAD INJECT
Measuring pump
28
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Column Oven
Air circulation heating type
Block heating type
Aluminum block heater
Insulated column jacket type
Water bath
29
30
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Tubing
Material
Stainless steel (SUS) O.D. (outer diameter)
PEEK (polyether ether 1.6 mm
ketone) I.D. (inner diameter)
Fluororesin 0.1 mm
0.3 mm
0.5 mm
0.8 mm etc.
31
Connectors
32
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Dead Volume
(Extra-column volume)
Tube
Mobile Phase
Water Organic Solvent
Ultrapure water can be HPLC-grade solvent can be
used with confidence. used with confidence.
Commercial distilled water Special-grade solvent is
for HPLC is also acceptable. acceptable depending on the
detection conditions.
Care is required regarding
solvents containing stabilizers
(e.g., tetrahydrofuran and
chloroform)
34
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Replacement of Eluent
Aqueous solutions containing salt
Mutually insoluble solvents must and organic solvents must not be
not be exchanged directly. exchanged directly.
2-Propanol Water
Hexane Water-soluble
35 organic solvent
Decompression
by aspirator
Ultrasonic
cleaning unit
36
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37
Polarity of Substances
H
H O
O
C H C C
H H O
H + H
H
H
Methane Water
38 Acetic acid
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39
Partition Chromatography
40
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Stationary
Mobile phase
phase
Normal High polarity Low polarity
phase (hydrophilic) (hydrophobic)
41
42
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C18 (ODS)
43
C8
Medium
C18 (ODS)
Strong C4
Weak
44
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Hydrophobic Interaction
H2O H2O
H2O
H2O H2O the nonpolar substance
H2O H2O is pushed to a nonpolar
location.
Nonpolar solute
Nonpolar stationary
45 phase
C18 (ODS) OH
Weak
Strong
CH3
46
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47
CH3OH
Nonpolar solute CH3OH
Nonpolar solute
24
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60/40
70/30
80/20
49
Chromatogram Parameters
50
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Retention Factor, k
t R t0
Strength of detector signal
tR
k
t0
t0
tR: Retention time
t0: Non-retention time
Time
51
2
t
N 16 R
W
2
t
5.54 R
H W1/ 2
W1/2
tR H
2
H1/2
2
W Area
52
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If the retention times are the If the peak width is the same,
same, the peak width is the retention time is longer
smaller for the one with the for the one with the larger
larger theoretical plate theoretical plate number.
number.
N: Large N: Small
N: Large
N: Small
53
Separation Factor, a
Separation factor: Ratio of ks of two peaks
k1 k2 k2
k1
(k 2 k1 )
54
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Resolution, RS
tR1 tR2
t R 2 t R1
RS
1
(W1 W2 )
2
t R 2 t R1
1.18
W1/ 2 h ,1 W1/ 2 h , 2
W1/2h,1 W1/2h,2 h1/2
W1 W2
55
W1 W2 W1 W2
tR2 - tR1 = W1 = W2 tR2 - tR1 = W1 = W2
RS = 1 RS = 1
If the peaks are isosceles triangles, If the peaks are Gaussian distributions,
they are completely separated. RS > 1.5 is necessary for complete separation.
56
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The resolution is a
t t
function of the RS R 2 R1
separation factor, the 1
(W1 W2 )
theoretical plate number, 2
and the retention factor.
1 1 k2
The separation can be N
4 k2 1
improved by improving
these 3 parameters!
57
58
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The resolution
59
To Improve Separation...
Before
adjustment
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61
H+ If an acid is added...
HA A - + H+
If an alkali is
added...
The equilibrium always shifts
the equilibrium shifts to in a way that offsets changes.
the right to offset the OH-
decrease in H+.
62
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Abundance ratio
is defined as follows:
0,6
[A ][H ]
Ka 0,4
[HA]
[A ] 0,2
pH pK a log
[HA] 0,0
1 2 3 4 5 6 7 8 9
pH
pH log[ H ]
pKa
pK log K Relationship Between Abundance Ratio
a a and pH Value of Acetic Acid and Acetic Acid Ions
63
64
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65
66
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67
Eluent Composition
Water / acetonitrile
Water / methanol
Separation Adjustment
Changing the mixing ratio of the water and organic
solvent
Changing the type of organic solvent
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COOH
Acidic
Increased
hydrophobicity
pH of eluent
COO
Alkaline
decreased
hydrophilicity
+
H
69
Eluent Composition
Acidic buffer solution / acetonitrile
Acidic buffer solution / methanol
70
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OH Si
O
OH Si OH
silica gel dissolves in alkalis, so
the packing material deteriorates
OH rapidly.
OH
OH
71
Si
O
Si -O-Si-O
Residual silanol group N+
O H
Si
72
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Si
O
73
Eluent Composition
Acidic buffer solution containing anions with a low
charge density (e.g., perchlorate ions) / acetonitrile
As above / methanol
Making eluent acidic
Suppresses dissociation of residual silanol groups
Prevents tailing!
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76
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77
78
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79
Adsorption Chromatography
A solid such as silica gel is used as the
stationary phase, and differences, mainly in
the degree of adsorption to its surface, are
used to separate the solutes.
Liquid-solid chromatography
The retention strength increases with the
hydrophilicity of the solute.
80
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Partition Chromatography
81
82
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83
Invention of Chromatography by M.
Tswett
Ether
Chromatography
Chlorophyll Colors
CaCO3
84
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Stationary Phase
Silica gel: -Si-OH
Cyano type: -Si-CH2CH2CH2CN
Amino type: -Si-CH2CH2CH2NH2
Diol type: -Si-CH2CH2CH2OCH(OH)-CH2OH
Mobile Phase
Basic solvents: Aliphatic hydrocarbons,
aromatic hydrocarbons, etc.
Additional solvents: Alcohols, ethers, etc.
85
SiOH HO
Strong
SiOH
Weak
Very weak
OH
Steric hindrance
86
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Eluent: Hexane/methanol
100/0
98/2
95/5
87
88
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R
Anion exchange N+ R
R
++++
Cation exchange SO3- + +
++++
Electrostatic interaction
(Coulomb force)
89
90
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Exchange capacity
Weakly acidic
cation exchanger Weakly basic anion
exchanger
0 7 14 0 7 14
pH pH
Cation exchange mode Anion exchange mode
91
Solute ions and eluent ions compete for ion exchange groups.
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H+
H+
H+
Strong acid ions are repelled by charge Depending on the level of dissociation, some
and cannot enter the pore. weak acid ions can enter the pore.
93
94
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Packing
material
95
Exclusion limit
Molecular weight
(logarithmic axis)
Permeability limit
Elution capacity
96
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Elution capacity
For separation of small molecular weights
97
Chromatogram
Various Average Molecular
Weights
Mn: Number-average
molecular weight
Mw: Weight-average
Calibration curve molecular weight
Mz: Z-average molecular
weight, etc.
Molecular weights and
molecular weight distributions
are calculated using special
calculation software.
Retention time
98
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Soluble solvent
Molecular weight
Structural formula and chemical properties
Do the substances ionize?
Is there UV absorption or fluorescence?
Is derivatization possible? etc.
99
100
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101
102
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103
C: Concentration
Detection cell
Ein Eout
A
104
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Reference cell
D2 / W lamp
105
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Sample cell
Grating
A single photodiode
D2 / W lamp measures the absorbance for
the corresponding wavelength
at a resolution of approx. 1 nm.
Photodiode array
107
Chromatogram
Absorbance
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109
Fluorescence Detector
Excitation wavelength
+ hv1 *
* hv2 +
Fluorescence wavelength
Excited state
Quasi-excited state
hv1
hv2
Fluorescence
110
Ground state
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Xenon lamp
Fluorescence
grating
Photomultiplier tube
Fluorescence
Excitation
Excitation grating Sample cell
light
111
CHN2 CH2OCOR
9-anthryldiazomethane
(ADAM)
112
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Light-receiving unit
Reference cell
Light
Sample cell
113
Slit W lamp
Reference cell
Sample cell
The slit image moves if the
refractive index inside the
flow cell changes.
Photodiode
114
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Light-receiving unit
Drift tube
Nebulizer
Column eluate
Nebulizer gas
Drain Assist gas
Light source
115
Cl- Na+
The bulb does not light with water. The bulb lights if there are ions.
116
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I A
V K k
I E L
L
k K
A
A A K: Electrical conductivity [S]
I: Electric current [A]
E: Voltage [V]
A: Electrode surface area [cm2]
L Electrode L: Distance between electrodes [cm]
k: Specific electrical conductivity [Scm-1]
117
Cation l Anion l
H+ 349.8 OH 198.3
Li+ 38.6 F 55.4
Na+ 50.1 Cl 76.3
K+ 73.5 Br 78.1
NH4+ 73.5 NO3 71.4
(CH3)3NH+ 47.2 CH3COO 40.9
Mg2+ 53.0 C6H5COO 32.3
Ca2+ 59.5 SO42 80.0
118
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Electrochemical Detector
Electrode
HO R
HO
2e-
O R
+ 2H+
O
119
Eluent
Electrode couple
120
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Atmospheric
pressure High vacuum
Quadrupole MS analyzer
API probe
Electron
multiplier tube
RP TMP1 TMP2
(high vacuum pumps)
121
Liquid
LiquidSamples
Sample
1)
+
2) of S
Io
Co va
Ch
ap ve
on ra
ge
ol nt
E x tio
d
at
D
cl n
io
ro
us
n
pl
io
et
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m/z=150
C D
123
M/Z
M/Z
M/Z 124
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Comparison of Detectors
Possibility of
Selectivity Sensitivity
Gradient System
Light-absorbing
Absorbance ng Possible
substances
Differential
None g Impossible
refractive index
Evaporative light
Nonvolatile substances g Possible
scattering
Electrical
Ionic substances ng Partially possible
conductivity
Oxidizing / reducing
Electrochemical pg Partially possible
substances
Note: The above table indicates general characteristics. There are exceptions. 125
Post-Column Derivatization
Reaction
chamber
Pump
Reaction
solution
126
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127
Quantitative Analysis
128
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Qualitative Analysis
Identification based on retention time
Acquisition of spectra with detector
UV spectra
MS spectra
Transfer to other analytical instruments after
preparative separation
129
Quantitative Analysis
Quantitation performed with peak area or
height.
Calibration curve created beforehand using a
standard.
Absolute calibration curve method
Internal standard method
Standard addition method
130
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A2 A3
Peak area
C2
A2
A3
C3
A1
A4
C4 C1 C2 C3 C4
131 Concentration
Target Internal
substance standard A1 AIS Calibration curve
C1 CIS A4 /AIS
A2 AIS A3 /AIS
C2 CIS
A2 /AIS
A3 AIS
C3 CIS
A1/AIS
A4 AIS
C4 CIS C1/CIS C2 /CIS C3 /CIS C4 /CIS
Concentration of target substance /
132
Concentration of internal standard
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Same area
ratio
IS
X
9 L
injected
CX / CIS
133
100%
recovery
rate
Same area
ratio
IS
90% X
recovery
rate
CX / CIS
134
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135
Sample Pretreatment
136
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Objectives of Pretreatment
To improve the accuracy of quantitative values
To improve sensitivity and selectivity
To protect and prevent the deterioration of
columns and analytical instruments
To simplify measurement operations and
procedures
To stabilize target substances
137
138
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139
Deproteinization
Precipitation
Addition of organic solvent (e.g., acetonitrile)
Addition of acid (e.g., trichloroacetic acid,
perchloric acid)
Addition of heavy metal or neutral salt
Ultrafiltration
140
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Solvent with
high elution
strength
Target
component
Unwanted
components
141
Pre-Column Derivatization
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143
144
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Accuracy / Trueness
Measurement
Precision
Repeatability / Intra-Assay
Definition Precision
Degree of coincidence of Precision of measurements
taken over a short time
series of measurements
period under the same
obtained by repeatedly conditions
analyzing multiple samples
taken from a homogenous Intermediate Precision
test substance Reproducibility
Variance, standard
deviation, or relative
standard deviation of
measurements
146
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Specificity
147
Detection Limit
148
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Quantitation Limit
149
Linearity
Definition Evaluation Method
The ability of the analytical Samples containing different
method to produce quantities of the target
measurements for the substance (usually 5
quantity of a target substance concentrations) are analyzed
that satisfy a linear repeatedly, and regression
relationship. equations and correlation
Values produced by coefficients are obtained.
converting quantities or Residuals obtained from the
measurements of the target regression equations of the
substance using a precisely measurements are plotted,
defined formula may be used. and it is confirmed that there
is no specific slope.
150
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Range
151
Robustness
152
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Maintenance of Separation
Column
Extending the Columns Service Life
153
Silica-Based Resin-Based
Generally a wide
pH range 2 - 7.5
range
Organic Significant
No restrictions
solvent restrictions
Pressure Low pressure
25 MPa max.
resistance resistance
Depends on
Temperature 60C max.
packing material
154
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155
156
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158
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159
Guard column
Pre-column
160
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Column Rinsing
Use an eluent with a high elution capacity
Reversed phase mode: Solution with a high proportion of
organic solvent
Ion exchange mode: Solution with a high salt concentration
Consider secondary retention effects
To remove basic substances from a reversed phase column
Use an acidic solution and add an ion pair reagent.
To remove hydrophobic substances from an ion exchange
column Add an organic solvent.
161
2
t
N 16 R
W
2
t
5.54 R
H W1/ 2
W1/2
tR H
2
H1/2
2
W Area
162
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Column Storage
163
82