High Performance Liquid Chromatography (HPLC))

 pemisahan senyawa berdasarkan perbedaan

interaksinya dalam fase diam dan fase gerak
 merupakan automatisasi dari Liquid
Chromatography (LC) pada kondisi tertentu
untuk memperbaiki pemisahan analit dengan
waktu pemisahan yang singkat, volume analit
yang sangat sedikit pada diameter kolom yang
kecil dan tekanan tinggi.
 sesuai untuk pemisahan senyawa yang tidak
mudah menguap, tidak stabil secara thermal
dan memiliki berat molekul yang tinggi
Berdasarkan kepolaran:
 Polar ( Fase “Normal”):
Cyano : umum digunakan, kasar, polaritas menengah
Diol (-OH) : lebih polar dan retentive
Amino : kepolaran tinggi, kurang stabil
Silica : sangat kasar, murah, adsorbent

 Non-Polar (“Fase Terbalik”)

◦ C18, C8 : kasar, umum digunakan, lebih retentive
◦ C3, C4 : kurang retentive, umumnya digunakan untuk
peptida dan protein
◦ Fenil : lebih selektif daripada rantai alkil
◦ Amino : kurang retentive, bagus untuk pemisahan
karbohidrat
Bertindak sebagai pelarut yang baik untuk sampel
yang dianalisis

Ada dua tipe:

 Kromatografi fasa normal
Heksan ; diklorometane; isopropanol; metanol

Kepolaran meningkat

 Kromatografi fasa terbalik

air ; metanol, asetonitril, tetrahidrofuran (THF)
Kepolaran menurun
Komponen-komponen dalam HPLC

1. Wadah Pelarut
2. Degasser
3. Pompa
4. Sistem Injeksi Sampel / sample
injection port
5. Kolom
6. Detektor
7. Recorder
1. Wadah pelarut / solvent reservoir:
merupakan tempat fasa gerak.
2. Degasser
menghilangkan udara terlarut yang menginterferensi
proses check valve dan analisis
3. Pompa
Berfungsi memompa larutan (fasa gerak) ke aliran
kromatografi dengan laju alir tertentu
Ada dua tipe:
Isokratik : pompa mengirimkan komposisi fasa gerak
(eluen) yang konstan selama analisis.
Gradient : komposisi eluen dapat berubah selama
analisis.
4. Sistem injeksi sampel
Terdiri dari : manual dan autosampler

 manual injeksi dengan syringe, terdiri dari :

loop tertentu dengan variasi ukuran, syringe dengan
variasi ukuran.
  Fixed Loop Autosampler
◦ Loop diinstal pada valve dan dapat berubah untuk
volume injeksi yang berbeda.
◦ Eksternal syringe menarik dan mengisi loop

5. Kolom
tempat terjadinya pemisahan komponen-komponen
campuran karena perbedaan kekuatan interaksi
antara zat-zat terlarut terhadap fasa diam. Zat-zat
yang kuat interaksinya dengan fasa diam akan keluar
dari kolom terlebih dahulu.

Kolom biasa ditempatkan dalam oven. Mengapa?

Mengapa menggunakan kolom oven?
 Waktu retensi berkurang dan memungkinkan
untuk menaikkan laju alir
2.1 mm ID Column, 0.35 mL/min, 50/50 MeOH/Water

50°
C
45°
C

40°
C
35°
C

30°
C

25°
C
20°
C
SHIMADZU
Solutions for Science Since 1875
6. Detektor
UV-VIS:
Diode Array
Refractive Index
Fluorescence
Conductivity
Mass Spectrometer
karakteristik detektor untuk HPLC
 sensivitasnya tinggi
 respon yang menyeluruh terhadap sampel
 tidak meruska sampel
 tidak sensitif terhadap perubahan temperatur dan kecepatan
aliran fasa mobile
 dapat beroperasi secara terus menerus.
Advantages using HPLC : Disadvantages :
1. Needs a small 1. Need a skill to run the
sample with a high instruments
accuracy and precis
2. Solvents consuming
2. Non-destructed
sample during
operation compared
to GC.
 Column Parameters Instrument Parameters

 Column Material  Temperature

 Deactivation  Flow
 Stationary Phase  Signal
 Coating Material  Sample Sensitivity
 Detector
 Sample Parameters

 Concentration
 Matrix
 Solvent Effect
 Sample Effect
 Fields in Which High Performance
Liquid Chromatography Is Used

 Biogenic substances  Food products

 Sugars, lipids, nucleic  Vitamins, food additives,
acids, amino acids, sugars, organic acids,
amino acids, etc.
proteins, peptides, steroids,
amines, etc.  Environmental
 Medical products samples
 Inorganic ions
 Drugs, antibiotics, etc.
 Hazardous organic
substances, etc.
 Organic industrial
products
 Synthetic polymers,
additives, surfactants, etc.
LAAQ-B-LC001B 16
Predict the order of elution from first to last of the
following morphinane compounds from an ODS
column in an acetonitrile/buffer mixture pH 8 (10 : 90).
 Chromatogram Parameters

Methods for Expressing Separation

and Column Performance

LAAQ-B-LC001B 19
 Strength of detector signal Retention Factor, k

tR t R  t0
k
t0
t0
tR: Retention time
t0: Non-retention time

Time

the ratio of times that the solute remains in the stationary and
LAAQ-B-LC001B 20
mobile phases retention factor
 Retention Factor, k

 If the k is 1 : the solute remains in the

stationary and mobile phases for the same
time.
 If k is less than 1 : the solute is not retained
to a significant degree before elution.
 If k is 3 or greater : the solute undergoes
significant interaction with the stationary
phase before elution.
LAAQ-B-LC001B 21
 Calculation of t0

For example, if the inner diameter and length

of the column are 0.46 cm and 15 cm
respectively, the porosity is 0.6, and the
eluent flow rate is 0.8 mL/min, then t0 can be
calculated as follows:

t0 = (0.232    15)  0.6  0.8 = 1.87 [min]

LAAQ-B-LC001B 22
 Theoretical Plate Number, N

2
tR
N  16
W
2
tR
 5.54
H W1/ 2
W1/2
tR  H
2
H1/2
 2
W Area
LAAQ-B-LC001B 23
 “plate theory” model is based on the concept of handling the process of
chromatography as repeated solvent extraction in a flask.
“theoretical plate number” is the number of plates corresponding to the
extraction performed by the separation column.

If the theoretical plate number is large, this means that extraction is

performed a correspondingly large number of times, and indicates a
relatively high level of separation performance.

the theoretical plate number is an indicator of the

efficiency (performance) of the separation column.

LAAQ-B-LC001B 24
 Evaluation of Column Efficiency Based on
Theoretical Plate Number

 If the retention times are  If the peak width is the

the same, the peak width same, the retention time is
is smaller for the one with longer for the one with the
the larger theoretical plate larger theoretical plate
number. number.
N: Large N: Small
N: Large

N: Small

LAAQ-B-LC001B 25
 Separation Factor, a

 Separation factor: Ratio of k’s of two peaks

k1 k2 k2

k1
(k 2  k1 )

parameter used to describe how well two solutes are separated by a chromatographic
system: A value of a α ≥1.1 is usually indicative of a good separation
LAAQ-B-LC001B 26
 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
LAAQ-B-LC001B 27
resolution (RS) – resolution between two peaks is a second measure of how well two
peaks are separated:
tr2 – tr1
RS =
(Wb2 + Wb1)/2
where:
tr1, Wb1 = retention time and baseline width for the
first eluting peak
tr2, Wb2 = retention time and baseline width for the
second eluting peak

Rs is preferred over  since both

retention (tr) and column efficiency
(Wb) are considered in defining
peak separation.

Rs $ 1.5 represents baseline

resolution, or complete separation
of two neighboring solutes  ideal
case.

Rs $ 1.0 considered adequate for

most separations.
 Resolution Required for Complete
Separation

(tR2 - tR1) (tR2 - tR1)

W1 W 2 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.
LAAQ-B-LC001B 29
 Relationship Between Resolution
and Other Parameters

 The resolution is a
tR 2  tR1
function of the RS 
1
separation factor, the
(W1  W2 )
theoretical plate 2
number, and the
1  1 k’2
retention factor.  N
4  k’2  1
 The separation can be
improved by improving
these 3 parameters!

LAAQ-B-LC001B 30
 Contribution of Capacity Factor to
Resolution

 Increasing the capacity 1.0

Contribution ratio for resolution

factor improves 0.8
separation!
0.6
 A capacity factor of
around 3 to 10 is 0.4

appropriate. Exceeding 0.2

this just increases the 0.0
analysis time. 0 5 10 15 20
Capacity factor

LAAQ-B-LC001B 31
 Contribution of Theoretical Plate
Number to Resolution

 The resolution

Contribution factor for resolution

2,0
increases in
proportion to the
square root of the 1,0
theoretical plate
number.
0,0
0 10000 20000 30000
Theoretical plate number

LAAQ-B-LC001B 32
 To Improve Separation...

Before
adjustment

k’ increased Eluent replaced with one

of lower elution strength.

Column replaced with one of

N increased superior performance.
Column lengthened.

Column (packing material) replaced.

 increased Eluent composition changed.
Column temperature changed.
LAAQ-B-LC001B 33
Theory of Chromatography

1.) Typical response obtained by chromatography (i.e., a chromatogram):

chromatogram - concentration versus elution time

Wh

Wb

Inject

Where:
tR = retention time
tM = void time
Wb = baseline width of the peak in time units
Wh = half-height width of the peak in time units
Note: The separation of solutes in chromatography depends on two factors:

(a) a difference in the retention of solutes (i.e., a difference in their time or volume of
elution
(b) a sufficiently narrow width of the solute peaks (i.e, good efficiency for the separation
system)

Peak width & peak position

determine separation of peaks

A similar plot can be made in terms of elution volume instead of elution time. If volumes
are used, the volume of the mobile phase that it takes to elute a peak off of the column is
referred to as the retention volume (VR) and the amount of mobile phase that it takes to
elute a non-retained component is referred to as the void volume (VM).
2.) Solute Retention:

A solute’s retention time or retention volume in chromatography is directly related to the

strength of the solute’s interaction with the mobile and stationary phases.

Retention on a given column pertain to the particulars of that system:

- size of the column
- flow rate of the mobile phase

Capacity factor (k’): more universal measure of retention, determined from tR or VR.

k’ = (tR –tM)/tM

or

k’ = (VR –VM)/VM

capacity factor is useful for comparing results obtained on different systems since it is
independent on column length and flow-rate.
The value of the capacity factor is useful in understanding the retention mechanisms for a
solute, since the fundamental definition of k’ is:
moles Astationary phase
k’ =
moles Amobile phase

k’ is directly related to the strength of the interaction between a solute with the stationary
and mobile phases.

Moles Astationary phase and moles Amobile phase represents the amount of solute present in each
phase at equilibrium.

Equilibrium is achieved or approached at the center of a chromatographic peak.

When k' is # 1.0, separation is poor

When k' is > 30, separation is slow
When k' is = 2-10, separation is optimum
A simple example relating k’ to the interactions of a solute in a column is illustrated for
partition chromatography:

KD
A (mobile phase) A (stationary phase)

where: KD = equilibrium constant for the distribution of A between the mobile

phase and stationary phase

Assuming local equilibrium at the center of the chromatographic peak:

[A]stationary phase Volumestationary phase

k’ =
[A]mobile phase Volumemobile phase

Volumestationary phase
k’ = KD
Volumemobile phase

As KD increases, interaction of the solute with the stationary phase becomes more
favorable and the solute’s retention (k’) increases
 Volumestationary phase
k’ = KD
Volumemobile phase

Separation between two solutes requires different KD’s for their

interactions with the mobile and stationary phases

since DG = -RT ln KD
peak separation also represents different changes in free energy
3.) Efficiency:

Efficiency is related experimentally to a solute’s peak width.

- an efficient system will produce narrow peaks
- narrow peaks  smaller difference in interactions in order to separate two solutes

Efficiency is related theoretically to the various kinetic processes that are involved in
solute retention and transport in the column
- determine the width or standard deviation (s) of peaks

Estimate s from peak widths,

assuming Gaussian shaped peak:
Wh
Wb = 4s

Wh = 2.354s

Dependent on the amount of time that a solute spends in the column (k’ or tR)
Number of theoretical plates (N): compare efficiencies of a system for solutes that have
different retention times

N = (tR/s)2

or for a Gaussian shaped peak

N = 16 (tR/Wb)2

N = 5.54 (tR/Wh)2

The larger the value of N is for a column, the better the column will be able to separate
two compounds.
- the better the ability to resolve solutes that have small differences in retention
- N is independent of solute retention
- N is dependent on the length of the column
Plate height or height equivalent of a theoretical plate (H or HETP): compare efficiencies of
columns with different lengths:

H = L/N

where: L = column length

N = number of theoretical plates for the column

Note: H simply gives the length of the column that corresponds to one theoretical plate

H can be also used to relate various chromatographic parameters (e.g., flow rate, particle size,
etc.) to the kinetic processes that give rise to peak broadening:

Why Do Bands Spread?

a. Eddy diffusion
b. Mobile phase mass transfer
c. Stagnant mobile phase mass transfer
d. Stationary phase mass transfer
e. Longitudinal diffusion
a.) Eddy diffusion – a process that leads to peak (band) broadening due to the presence
of multiple flow paths through a packed column.

As solute molecules travel through the column,

some arrive at the end sooner then others simply
due to the different path traveled around the
support particles in the column that result in
different travel distances.

Longer path arrives at end of column after (1).

b.) Mobile phase mass transfer – a process of peak broadening caused by the
presence of different flow profile within channels or
between particles of the support in the column.

A solute in the center of the channel

moves more quickly than solute at the
edges, it will tend to reach the end of
the channel first leading to band-
broadening

The degree of band-broadening due to eddy diffusion and mobile phase

mass transfer depends mainly on:

1) the size of the packing material

2) the diffusion rate of the solute
c.) Stagnant mobile phase mass transfer – band-broadening due to differences in the
rate of diffusion of the solute molecules between the
mobile phase outside the pores of the support
(flowing mobile phase) to the mobile phase within
the pores of the support (stagnant mobile phase).

Since a solute does not travel down

the column when it is in the stagnant
mobile phase, it spends a longer time
in the column than solute that
remains in the flowing mobile phase.

The degree of band-broadening due to stagnant mobile phase mass

transfer depends on:

1) the size, shape and pore structure of the packing material

2) the diffusion and retention of the solute
3) the flow-rate of the solute through the column
d.) Stationary phase mass transfer – band-broadening due to the movement of solute
between the stagnant phase and the stationary phase.

Since different solute molecules

spend different lengths of time in the
stationary phase, they also spend
different amounts of time on the
column, giving rise to band-
broadening.

The degree of band-broadening due to stationary phase mass transfer

depends on:

1) the retention and diffusion of the solute

2) the flow-rate of the solute through the column
3) the kinetics of interaction between the solute and the
stationary phase
e.) Longitudinal diffusion – band-broadening due to the diffusion of the solute along the
length of the column in the flowing mobile phase.

The degree of band-broadening due

to longitudinal diffusion depends on:

1) the diffusion of the solute

2) the flow-rate of the solute through
the column
Van Deemter equation: relates flow-rate or linear velocity to H:

H = A + B/m + Cm

where:

m = linear velocity (flow-rate x Vm/L)

H = total plate height of the column
A = constant representing eddy diffusion &
mobile phase mass transfer
B = constant representing longitudinal diffusion
C = constant representing stagnant mobile phase
& stationary phase mass transfer

One use of plate height (H) is to relate these kinetic process to band broadening to a
parameter of the chromatographic system (e.g., flow-rate).

This relationship is used to predict what the resulting effect would be of varying this
parameter on the overall efficiency of the chromatographic system.

Number of theoretical plates(N) (N) = 5.54 (tR/Wh)2 peak width (Wh)

H = L/N
Plot of van Deemter equation shows how H changes with the linear velocity (flow-rate) of
the mobile phase

m optimum

Optimum linear velocity (mopt) - where H has a minimum value and the point of maximum
column efficiency:

mopt = rB/C

mopt is easy to achieve for gas chromatography, but is usually too small for liquid
chromatography requiring flow-rates higher than optimal to separate compounds
4.) Measures of Solute Separation:

separation factor () – parameter used to describe how well two solutes are separated by
a chromatographic system:

 = k’2/k’1 k’ = (tR –tM)/tM

where:
k’1 = the capacity factor of the first solute
k’2 = the capacity factor of the second solute,
with k’2 $ k’1

A value of  $1.1 is usually indicative of a good separation

Does not consider the effect of column efficiency or peak widths, only retention.