High Performance Liquid

Chromatography
Kromatografi Cair Kinerja Tinggi

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 HPLC
C
P I

FD

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aliran fase gerak
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4. Komponen dideteksi oleh detektor
5. Data diproses oleh komputer
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 Applications

• The combination of high-pressure liquid chromatography (HPLC) with

monitoring by UV/ visible detection provides an accurate, precise and
robust method for quantitative analysis of pharmaceutical products
and is the industry standard method for this purpose
• Monitoring of the stability of pure drug substances and in drugs in
formulations with quantitation of any degradation products.
• Measurement of drugs and their metabolites in biological fluids.
• Determination of partition coefficients and pKa values of drugs and of
drug protein binding.

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 Strengths

• Easily controlled and precise sample introduction ensures quantitative

precision.
• HPLC is the chromatographic technique which has seen the most
intensive development in recent years leading to improved, columns,
detectors and software control
• The variety of columns and detectors means that the selectivity of the
method can be readily adjusted.
• Compared to GC there is less risk of sample degradation because
heating is not required in the chromatographic process
• Readily automated.

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 Limitations

• There is still a requirement for reliable and

inexpensive detectors which can monitor
compounds that lack a chromophore
• Drugs have to be extracted from their
formulations prior to analysis
• Large amounts of organic solvent waste is
generated, which is expensive to dispose of

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 Modes of HPLC
• Normal Phase mode
• Reverse Phase mode
• Reverse Phase Ion Pairing mode
• Ion Exchange mode
• SEC mode ( GPC / GFC )
• Chiral separation mode

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 Reverse Phase HPLC Columns
• C18 (ODS) type
• C8 (octyl) type
• C4 (butyl) type Non-polar property
• Phenyl type -Si-C18H35
• TMS type
• Cyano type Si

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 Structural Factors Which Govern Rate of
Elution 0f Compounds from HPLC Columns
• Elution of neutral compounds
– For a neutral compound it is the balance between its polarity and Iipophilicity
which will determine the time it takes for it to elute from an HPLC column; the
pH of the mobile phase does not play apart. In the case of a reverse-phase
column, the more lipophilic a compound is the more it will be retained
►
• Ionizable compounds
– Control of elution rate of ionisable compounds by adjustment of pH of mobile
phase. Untuk asam K’app = K’/(1+10pH-pKa) untuk basa K’app = K’/(1+10pKa-pH) ►
– Mobile-phase conditions may be selected in straight-phase chromatography
where the ionisation of the analytes is suppressed, and basic compounds are run
in a basic mobile phase and acidic compounds are run with an acidic mobile
phase
– The pH of the mobile phase can only be set within the range of ca 2-8.5 pH
units because of the tendency for extremes of pH to dissolve silica gel and
break the bonds between silane-coating agents and the silica gel support

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 Hydrophobicity

• If the sample has

– CH3CH2CH2--- : Carbon chain Hydrophobicity
– : Aromatic group becomes strong.

• If the sample has

-COOH : Carboxyl group
-NH2 : Amino group Hydrophobicity
-OH : Hydroxyl becomes weak.
group
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Retention Time and Hydrophobicity

OH

C18 (ODS)
Weak

Strong
OH

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 Increase of solvent polarity
20 % 30 % 40% / H2O

1 : p-Hydoxymethylbenzoate
2 : p-Hydoxyethylbenzoate Solvent : MeOH
3 : p-Hydoxypropylbenzoate
4 : p-Hydoxybutylbenzoate 11
Effect of stationary phase

C8

Medium
C18 (ODS)
sample
Strong C4
sample
Weak
sample

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 Effect of stationary phase

 Analytical Conditions
ODS C8 TMS  Column : Shim-pack CLC-ODS
 Mobile phase : MeOH : H2O = 7 :3
 Flow rate : 1.0 mL/min
 Temperature : 40 C
 Injection volume : 10 uL
 Detection : UV-254 nm
 Peaks
1. Methyl benzoate
2. Ethyl benzoate
3. n-Propyl benzoate
4. n- Butyl benzoate

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 Reverse Phase
Ion-Pair Chromatography

Ion-Pair Reagent

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 Ion Paring
Important Considerations
• Type of Ion-Pair reagents
• Concentration of Ion-Pair reagents
• pH of solvent

R-COOH RCOO- + H+
(pKa=4.5)
R-NH2 + H+ R-NH3+
(pKa=6.0)
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 Type of Ion-Pair Reagents
Hexane Sulfonate Pentane Sulfonate

Mobile Phase: H2O/MeOH

1:1,with 0.005M ion pairing
reagent and 1% HOAc

1 Maleic Acid
2 Phenylephrine
3 Phenylpropanolamine
4 Naphazoline
5 Phenacetin
6 Pyrilamine

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 Concentration of
Ion-Pair Reagents

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 Causes of Tailing Peaks
• Built-up of garbage on the column inlet
• Extra column effects (dead volume)
• Sample Overload
• Incorrect solvents for the sample
• Secondary retention effects
– Silanol group
– Residual heavy metal

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 Dead Volume

• Dead volume will cause a broad peak.

– Sample Injector Connection
– Column Connection
– Detector Connection
Good

Dead Volume
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 Incorrect solvent for sample
• Better do not select a high soluble solvent as a sample
solvent.
Methanol as a sample solvent Ethanol as a sample solvent

20 uL Caffeine 20 uL Caffeine

Ethanol as a sample solvent

Better inject small
10 uL Caffeine amount of sample.

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 Solubility effect

Low soluble solvent High soluble solvent

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 Secondary retention effects

• Silanol Group
– Even modified silica gel (e.g. ODS, C8), residual
silanol group are still remained on the surface
area.
– Silanol group will strongly absorb amine
compounds, therefore tailing will be happened.
C18
OH
Silanol group
silica core C18
O- negative charge
C18 22
C18
 End capping

• To suppress the silanol group effect, an end

capping type of column will be selected.

C18 C18
OH TMS treatment O-TMS
silica core C18 silica core C18
OH TMS : trimethylsilyl group
O-TMS
C18 C18
C18 C18
[Non-End capping type] [End capping type]
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 Secondary retention effects

• Residual heavy metal

– Normal silica gel has heavy metal as an impurity.
This heavy metal will have an interaction with
chelate compounds, therefore tailing will be
happened.

C18
M+ O-TMS High pure type of
silica core C18 O
+ O-TMS silica gel are available.
M C18 O
C18
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 HPLC system

• Isocratic elution system

– Single solvent of constant composition
• Gradient elution system
– Multi solvents of changeable composition
• High pressure gradient system
• Low pressure gradient system

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Isocratic Elution System

pump injector oven

detector

column

Single Solvent
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Gradient Elution System

A
column
injector detector
pump oven

B concentration
B

pump

Time
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 Isocratic Elution Mode

MeOH / H2O = 6 / 4
Long Time Analysis

Bad Separation

MeOH / H2O = 8 / 2

( column : ODS type ) 28

 Gradient Elution Mode

MeOH concentration
95%

30%

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 Calibration method

• External calibration method

• Internal calibration method
• Standard additive method
• Correction Normalization method

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 External Standard Calibration
Preparation of Standards

Target Compounds

Dilution Dilution Dilution Dilution

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 External Standard Calibration

200
180
Concentration

160
140
120
100
80
60
40
20
0
0 1000 2000 3000 4000
Peak Area

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 External Standard Calibration
Calculation of Results

Y = bX + a
[Concentration]

b : SLOPE
125 ppm a : Y intercept

2500
2500
[Peak Area]

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 Internal Standard Calibration
Preparation of Standards

Internal
Target Compounds Standard

Dilution Dilution Dilution Dilution

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Internal Standard Calibration
Analysis of Vanillin

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 Internal Standard Calibration
[Terget Conc. / IS Conc.]
Analysis of Vanillin

4
3.5
3
2.5
2
1.5
1
0.5
0
0 5 10 15
[Tergat Area / IS Area]

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 Internal Standard Calibration
Calculation of Results
[Target Conc. / IS Conc.]

Y = bX + a
b : SLOP
1.67 a : Y intercept

T 2500
5.0
500
[Target Area / IS Area] IS
Y = Target Conc. / IS Conc.
1.67 = Target Conc./ 100 ppm
Target Conc. = 167 ppm
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 Advantage of external standard
calibration method
• Only the target compound separation can be
focused.

Target Target

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 Disadvantage of external standard
calibration method
• Injection error will directly influence the
quantitative result.
10 uL injection 11 uL injection

100 ppm 110 ppm

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 Advantage of internal standard
calibration method
• Injection error can be eliminated.

10 uL injection 11 uL injection
2200
2000 1100
1000 T
IS
IS T

2000 / 1000 = 2 2200 / 1100 = 2

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 Advantage of internal standard
calibration method

• Recovery in the pretreatment process can be

estimated. addition of IS (100 ppm)
standard IS (100 ppm) to actual sample
IS T
IS T
1000 950

Recovery = (950/1000)x100 = 95%

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 Disadvantage of internal standard
calibration method
• Separation is slightly difficult.

IS T IS T

T
IS

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 Disadvantage of internal standard
calibration method

• It is difficult to look for the IS compound.

– The chemical structure of IS compound is
similar with one of target compound.
– IS sample is not existent in the actual sample.

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 Calibration Method
• External standard calibration
– Separation is not difficult
– Injection error will directly influence the
quantitative result
• Internal standard calibration
– Injection error can be eliminated
– Recovery in the pretreatment procedure can be
estimated
– Separation is slightly difficult
– Difficult to look for the IS compound

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 Standard additive
calibration method

Original
Sample T
Target

T
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 Standard additive
calibration method

T x104
Peak Area 100 ppm=10x104
17 ??? ppm= 7x104
T 12

7
??=70 ppm
T

-70 0 50 100 ppm

Added amount 46