PerkinElmer HPLC & UHPLC Workshop

HPLC & UHPLC
Workshop
PerkinElmer Korea
2009 9 18
© 2009 Perkin Elmer
HPLC
Basic Principle
PerkinElmer Korea
Kim, Wang-Yu PhD
© 2009 Perkin Elmer

Chromatography
Chromatography h
Chromos(color) + graphein(write) =
1906 Michael Tswett
.
(Liquid-Solid).
Molecular components in a mixture are separated because of
their affinities for two substances called phases.
()
3
HPLC
•
4

HPLC
Chromatogram
Injection
Port
computer
high
pressure
Pump
Colum mn
Detector
5
Chromatography
chromatography
Gas chromatography
Liquid Chromatography
Gas-Solid
Gas-Liquid
Adsorption Partition Ion exchange Size Exclusion Affinity
Gel Filtration
Gel Permeation
6

High Performance
Liquid Chromatography
7
Partitionchromatography
•
•
•
•
•
8

(reverse phase)
O
O
Si
O
O H +
Cl
CH 3 Si
(CH 2 ) 17
CH 3 CH 3
O
CH 3
O Si O Si (CH 2 ) 17 CH 3
O
CH 3
Pore
Si
Si
CH
3
Si -O-Si-(CH 2 ) - CH
17 3
Si CH 3
Si
Si
9
10

Adsorbent(
Solvent
Pentane
Hexane
Iso-octa
ne
Cyclohex xane
Carbon
tetrachlo
oride
l-Chlorob butane
Xylene
Toluene
Chlorobe enzene
Benzene
e
Ethyl eth her
Dichloro
methane
Chlorofo orm
1,2-Dich hloroethane
Methyl ethyl ketone
Acetone
Dioxane
1-Pentan nol
Tetrahyd drofuran
Methyl t
-butyl ether
Ethyl ace etate
Dimethy yl sulfoxide
Diethyla
mine
Acetonit
rile
1-Butano ol
Pyridine
2-Metho oxyethanol
n-Propyl
l alcohol
Isopropy yl alcohol
Ethanol
Methano ol
Ethylene e glycol
Dimethy yl
formami ide
Water
(Al 2
O 3
)
0.00
0.00-0.01
0.01
0.04
0.17-0.18
0.26-0.30
0.26
0.20-0.30
0.30-0.31
0.32
0.38
0.36-0.42
0.36-0.40
0.44-0.49
0.51
0.56-0.58
0.56-0.61
0.61
0.45-0.62
0.3-0.62
0.58-0.62
0.62-0.75
0.63
0.52-0.65
0.70
0.71
0.74
0.78-0.82
0.78-0.82
0.88
0.95
1.11
-
-
(SiOH)
0.00
0.00-0.01
0.01
0.03
0.11
0.20
-
0.22
0.23
0.25
0.38-0.43
0.32-0.32
0.26
-
-
0.47-0.53
0.49-0.51
-
0.53
0.48
0.38-0.48
-
-
0.50-0.52
-
-
-
-
0.60
-
0.70-0.73
-
-
-
(C 18
)
P
_ 0.0
_ 0.1
_ 0.1
_ 0.2
_ 1.6
_ 1.0
_ 2.5
_ 2.4
_ 2.7
_
_
_ 2.8
_ 3.1
_ 4.1
_ 3.5
_ 5.7
8.8 5.1
11.7 4.8
-
-
3.7 4.0
- 2.5
- 4.4
- 7.2
-
-
3.1 5.8
- 3.9
- 5.3
- 5.5
10.1 4.0
8.3 3.9
3.1
-
1.0 5.1
-
-
7.6 6.4
- 10.2
11
Chromatography
()
HPLC
HPLC
12
HPLC column

Isocratic vs Gradient
‣
‣
13
Polarity
Functional Group Polarity Comparisons
Polarity Functional Group Structure Bonding Types Intermolecular Forces Displayed
Low Methylene σ London
R (CH 2 ) 2
Phenyl σ , π London
R
Halide R F, Cl, Br, I
σ London, Dipole-Dipole
Ether
R O
σ London, Dipole-Dipole, H-bonding
R
O -
Nitro
R N + O
σ , π London, Dipole-Dipole, H-bonding
Ester R
O
σ , π London, Dipole-Dipole, H-bonding
O R
O
Aldehyde σ , π London, Dipole-Dipole, H-bonding
R
H
Ketone R
O
σ , π London, Dipole-Dipole, H-bonding
R
Amino R NH 2
σ , π London, Dipole-Dipole, H-bonding, Acid-base chemistry
Hydroxyl R OH
σ London, Dipole-Dipole, H-bonding
O
High Carboxylic Acid R
σ , π London, Dipole-Dipole H-bonding Acid-base OH
14

AbsorptionFluorescence
Absorption
Fluorescence
Luminescence
15
Ion exchange() chromatography
eletrostatic force
polystyrene or silica gel
16

Ion exchage column
Strong Cation Exchanger
Polystyrene
or
Silica
R1
(CH 2 CH 2 ) SO 3
-
R4 N +
R2
R3
Strong Anion Exchanger
Polystyrene
or
Silica
CH 2 CH 2 CH 2 NR 3
+ -
OOC R
Application :
17
Size exclusion() chromatography
18

Size exclusion
19
High Performance
Liquid Chromatography
20

Mass transfer( )
21
Longitudinal diffusion()
22

Eddy-diffusion()
Initial Band Width
Final
Band
Width
23
Van Deemter Equation
B
u
A
24

• Column: C18, 4.6 x 150 mm (5 µm)
• Mobile Phase: 82% H 2 O : 18% ACN
• Injection Volume: 20 µL
• Sample: 1. Caffeine
2. Salicylamide
RT(retention time)
• Column temperature : 30 degree
• Detection : 254 nm
INJECT
calibration :
Integration :
25
Chromatogram
RT(retention time)
R2
’ R1
’
R1
0
’ ’
0 R1 R2
α
’ R1
’ R2
INJECT
26

Selectivity α
R 2
0
’ R 2
’ R 1
α = =
R
1 0
α
27
Separation of water soluble vitamins
Mobile Phase: 15% MeOH
85% (10 mM hexanesulfonate,
Separations of Water-Soluble Vitamins
1% HOAc, 0.13% TEA in H 2 0)
using different tC8 and C18 columns Flow Rate: 1.5 mL/min at 35 C
1. Vitamin C
2. Niacin
3. Niacinamide
4. Pyridoxine
5. Thiamine
6. Folic Acid
7. Riboflavin
28
Ref.: M.W. Dong and J. L. Pace, LC.GC, 14(9), 794-803, 1996.

Efficiency
Inject
t R t N = 16 ( R
t
) 2 = 5.54 ( R
W
) 2
b
W 1/2
W 1/2 HETP = L/N
W b
•
•
• 29
N = 5000
N = 10,000
N = 20,000
1 3 5 7 9 11 13 15
Time (min)
30

Resolution(
R s =
1
√ N
4
α -1
α
k’
1 + k’
R = 0.4 R = 0.5 R = 0.6 R = 0.7
R = 08 0.8 R=10 1.0 R=15
1.5
31
32

HPLC
column & separation
PerkinElmer Korea
Kim, Wang-Yu PhD
© 2009 Perkin Elmer
HPLC
Chromatogram
Injection
Port
computer
high
pressure
Pump
Colum mn
Detector
34

35
• Short (30-50mm) - short run times, low backpressure
• Long (250-300mm) - higher resolution, long run times
• Narrow (≤ 2.1mm) - higher detector sensitivity
• Wide (10-22mm) - high sample loading
• Narrow columns enable to save solvent waste.
• Short columns enable to reduce the analyssis time.
36

4.6 mm
2.1 mm
L = 250 mm
L = 150 mm
37
• Spherical particles offer reduced back pressures and longer column life when
using viscous mobile phases like 50:50 MeOH:H2O.
38

• Smaller particles offer higher efficiency, but also cause higher backpressure.
Choose 3µm particles for resolving complex, multi-component samples.
Otherwise, choose 5 or 10µm packings.
P ∝ (1/d p ) 2 P = Pressure
d p = Particle Size
39
• High surface area generally provides greater retention, capacity and resolution
for separating complex, multi-component samples. Low surface area packings
generally equilibrate quickly, especially important in gradient analyses.
40

• Larger pores allow larger solute molecules to be retained longer
through h maximum exposure to the surface area of the particles.
Choose a pore size of 60~150Å or less for sample MW ≤ 2000.
Choose a pore size of 300Å or greater for sample MW > 2000.
41
Higher carbon loads generally offer greater resolution and longer
run times. Low carbon loads shorten run times and many show a
different selectivity.
42

Endcapping reduces peak-tailing of polar solutes that interact excessively with the
otherwise exposed, mostly acidic silanols. Non-endcapped packings provide a
different selectivity than do endcapped packings, especially for such polar
samples.
43
Brownlee(from PerkinElmer) Validated
44

Question : What column?
45
Answer :
C18
Phenyl
46

Question : What column?
Which two sample components have the most similar structures?
Draw them, then circle the structural t differences between them.
Anthracene
3-Hexylanthracene
(CH2) 5 CH 3
Note: The structural difference between
these two compounds is the
hydrophobic hexyl side chain. This
suggests a non-polar C18 or C8
column would interact with this area of
difference to help provide separation of
these two compounds.
Recommended bonded phase (silica based materials only) – mark one
Normal phase silica NH 2 CN
Reversed phase C18 C8 Ph CN
47
: Column problem
A.
: plugged frit, column contamination
B.
: split peak, peak tailing, broad peak
C.
: Equilibration, flow change, selectivity change
48

49
Column: C18, 4.6 x 150 mm, 5 µm Mobile Phase: 82% H 2 O : 18% ACN
Injection Volume: 30 µL Sample: 1. Caffeine 2. Salicylamide
A. Injection Solvent
B. Injection Solvent
100% Acetonitrile il
Mobile Phase
2
2
1
0 10
Time (min)
0 10
Time (min)
50

Mobile Phase: 40% 5 mM KH 2 PO 4 : 60% ACN
Flow Rate: 1.0 mL/min. Temperature: RT
.
pH 4.4 pH 3.0
CH 3 CHCOOH
CH 2 CH(CH 3 ) 2
Ibuprofen
pK a = 4.4
0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10
Time (min)
Time (min)
[RCOO - ][H + ]
RCOOH RCOO - + H + K a = [RCOOH]
51
May be caused by:
• Column aging
• Column contamination
• Insufficient equilibration
• Poor column/mobile phase combination
• Change in mobile phase
• Change in flow rate
• Other instrument issues
52

UHPLC
Basic principle
PerkinElmer Korea
Kim, Wang-Yu PhD
© 2009 Perkin Elmer
HPLC
Chromatogram
Injection
Port
computer
high
pressure
Pump
Colum mn
Detector
54

Chromatogram()
RT(retention time)
R2
R1
0
’ R1
INJECT
’ R2
55
Benefits of UHPLC
UHPLC
Green Productivity
• Higher throughput
• Much Less Solvent
• Better detection
Isoflavones in nutriceutical products
Can be analyzed with UHPLC over six times
faster, with higher sensitivity,
using over 90% less mobile
phase solvent compared with
conventional HPLC methods.
Conventional
HPLC
56
0 5 10 15 20 min.
…save time and money

Common Fears of Migrating to UHPLC
New Technology
Cost of new hardware
Cost of ownership and operation
Ruggedness of sub-2µm columns
High pressure
Training
i
Software
Method conversion
• Migration to UHPLC is easier than most people p think.
• New hardware will pay for itself by increasing productivity and
reducing operating costs.
57
Don’t Be Afraid of UHPLC!!!
Principles and Theory of HPLC
Resolution Equation
1 ⎡
k′
⎤
R s
= N • • −1
4 ⎢
⎥ α
⎣ 1 + k′
⎦
( )
Efficiency
• particle size
• column length
• solvent velocity
Retention
•solvent strength
th
• stationary phase
composition
Selectivity
• type of
stationary phase
• mobile phase
composition
• additives
58
…baseline separation is the goal for identification and quantification

UHPLC – High Resolution and Speed
1 ⎡ k′
⎤
= N
• •
4 ⎢
⎣ 1 + ′ ⎥ α
k ⎦
−
( 1
)
R s
N α 1 Efficiency is inversely
Efficiency Retention capacity Selectivity
N α proportional to particle
dp size (d p )
L Efficiency is directly
N α proportional to column
d dp
length (L)
59
…the strength of UHPLC is increased efficiency by reducing particle size
‘Van Deemter Equation’
Smaller particles size means
Faster chromatography
Increased Efficiency
Enhanced Resolution/Selectivity
Wider range of flow velocities
Less solvent consumption
Smaller particles size also means
Higher operating pressure
Filtration of MP and samples (0.2µm)
Low system volume required
Lower sample loading
Higher skill level of operator
60
…the benefits justify the challenge

Van Deemter Equation - Contributions to Band Broadening
H
H = A + B u + Cu
Van Deemter equation describes column
efficiency as a function of mobile phase Actual Plot
velocity in terms of solute:
• eddy diffussion i (A)
• molecular diffusion (B)
• mass transfer (C)
C Term
Optimum efficiency
A Term
B Term
Linear Velocity (u)
61
Van Deemter Equation - Contributions to Band Broadening
H = A + B u + Cu
Eddy Diffusion
H
2
3
Column
Faster
1
Time
Slower
A Term
• Independent d of mobile phase velocity (u)
• A decreases as particle size (d p ) decreses
Linear Velocity (u)
62

Van Deemter Equation - Contributions to Band Broadening
H = A + B u + Cu
Molecular Diffusion
H
• Describes diffusion of solute in the mobile phase
• Effect of B becomes negligible at sufficient flow rate
A Term
B Term
Linear Velocity (u)
63
Van Deemter Equation - Contributions to Band Broadening
H = A + B u + Cu
H
• Describes the transfer
of solute into and out
of the stationary
phase (sorption and
desorption).
• C decreases with
particle size (d p ) so
slope of C*u also
decreases with
particle size.
Mass Transfer
stationary silica
mobile phase
phase support
A Term
B Term
Linear Velocity (u)
64

Van Deemter Equation - Contributions to Band Broadening
H = A + B u + Cu
H
• Van Deemter equation terms A and C decrease as
particle size decreases.
• Sub-2µm columns are more efficient over a
broader range of flow rates.
Optimum efficiency
ATerm
Linear Velocity (u)
B Term
CTerm
A Term
B Term
65
UHPLC Basics
Sub-2µm Particle Columns
Increase Column Efficiency ( N ∝1
d p )
Improve Resolution ( R ∝ 1 )
s
d p
2
Increase Column Backpressure ( ∆P ∝ d )
1 p
150mmx46mmx50µm
4.6 x 5.0 50mmx21mmx19µm
2.1 x 1.9 • N ≈ Constant (~ 10,000 plates)
• R s ≈ Constant
t
• ∆P (~ 6X)
• t R (~ 8X)
66
…faster analysis at higher pressure

Requirements for a UHPLC System
System must handle pressures up to 15,000 psi to in order to utilize sub 2-µm
particle size columns.
• Ultra high pressure pumps such as the Flexar FX-10 and FX-15.
• Fast autosamplers with injection valves that can handle pressures >15,000 psi.
Column oven – elevated temperature reduces mobile phase viscosity and system
backpressure.
Fast detectors such as the Flexar FX UV/Vis with a sampling rate of 100 Hz.
Reduced extra-column volume is critical to minimize band broadening in UHPLC.
• Low ID Tubing
• Short tubing length
• Low volume detector
flow cell
Contributions of
Band Broadening
col
67
…Flexar FX UHPLC systems are up to the task
Extra column volume
Extra-Column-Volume =
sample volume + connecting tubing
volume
+ fitting volume + detector cell volume
Tubing volume
68

Band Broadening Comparison
85.00
80.00
75.00
1400
3360
Conventional system
IBW = 22 µL
70.00
65.00
60.00
100.00 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9
90.00
5700
80.00
3800
Optimized UHPLC system
IBW = 12 µL
70.0000
60.00
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9
69
…minimizing extra-column volume significantly reduces band broadening
General Steps of Method Migration
1. Choose a HPLC analysis that is appropriate for migration to UHPLC.
2. Select an UHPLC column with the best potential for successful migration.
3. Calculate UHPLC method parameters (Flow Rate, Injection Volume, etc.).
4. Run transferred method.
5. Optimize transferred method.
Other Considerations:
• Use only HPLC grade or UHPLC grade mobile phases.
• Filter all mobile phases, standards and samples with ≤0.2µm membranes.
• Utilize elevated temperature to reduce MP viscosity and column
backpressure.
• Prepare samples and standards in initial MP conditions.
70
…careful planning maximizes potential for success

Choosing the Right Column
Choose a column with the same type of stationary phase to maintain retention
order.
The optimal column ID for UHPLC is 2.1mm because mobile phase
consumption and frictional heating are significantly reduced.
Choose column dimensions that will have a similar efficiency as the HPLC
column currently used.
A good “rule of thumb” for choosing a UHPLC column length when currently
using a 4.6mm ID, 5.0µm particle HPLC column is:
HPLC Column Dimensions
50mmx46mm
4.6 UHPLC Column Dimensions
30 mm x 2.1 mm
150 mm x 4.6 mm
50 mm x 2.1 mm
250 mm x 4.6 mm
100 mm x 2.1 mm
71
…the right column choice will simplify method migration
Tools Available to Simplify Method Migration
Many good references are available with detailed descriptions of method transfer
including a PerkinElmer white paper entitled “Guidelines for the Use of UHPLC
Instruments” by D. Guillarme & J. Veuthey. White paper is available at:
http://las.perkinelmer.com/content/applicationnotes/wtp_guidelinesforuhplcinstruments.pdf
There are five major method
parameters to convert when
transferring a method from
HPLC to UHPLC.
• Injection Volume (V inj )
• Flow Rate (F)
• Isocratic Step Time (t iso )
• Gradient Step Time (t grad )
• Gradient Slope
72
…method transfer calculator is available from PerkinElmer

Injection Volume
The injection volume (V inj ) must be adjusted to avoid column overload as well as
maintain sensitivity and reduce extra-column band broadening.
As a rule, injected volumes should not exceed 1-5% of the column volume.
The column volume is a function of the column length (L) and internal diameter
(d c ) but is independent of stationary phase particle size.
V
2
dc
ij inj
=
Vij
inj
×
×
1
2
dc
L
2
2
L
1
2
1
V inj = Injection Volume
d c = Cl Column Diameter
L = Column Length
• 150 mm x 4.6 mm x 5.0 µm 50 mm x 2.1 mm x 1.9 µm
V inj1 = 15.0 µL V inj2 = 1.0 µL
15X
Decrease in Injection Volume
73
…conserve precious sample and expensive standards
Flow Rate
The flow rate (F) for UHPLC must be adjusted to maintain a similar mobile phase
linear velocity (u) used in the HPLC column.
The linear velocity within a column is directly proportional to the column diameter
(d c ) but also depends on the particle size (d p ) of the stationary phase. Therefore
u*d p must be maintained at a constant value to account for changes in column
diameter and particle size.
2
F =
F
×
×
2
1
2
d c
d p
1
2
d c
d p
1
2
F = Flow Rate
d c = Column Diameter
d p = Particle Diameter
• 150mmx46mmx50µm 4.6 x 5.0 50mmx21mmx19µm
2.1 x 1.9 F 1 = 10mL/min 1.0 F 2 = 054mL/min
0.54 ~2X
Decrease in Flow Rate
74
…save on mobile phase consumption

Isocratic Step Times
% B
The ratio between the isocratic
step time (t iso ) and the column
dead time must be maintained
between HPLC and UHPLC
conditions.
Time
The column dead time depends on the flow rate, column diameter and length.
t
2
F d
1 c L
2 2
iso
= t
2 iso
× × ×
1
2
F2
d L
c
1
2
1
F = Flow Rate
d c = Column Diameter
L = Column Length
• 150 mm x 4.6 mm x 5.0 µm 50 mm x 2.1 mm x 1.9 µm
t iso1 = 5.0 min
t iso2 = 0.6 min
75
5 min 0.6 min Isocratic Step
Gradient Slope
The initial and final MP compositions in
% B any HPLC gradient step should be
maintained in the UHPLC method.
Time
2
d
c
1
slope
2
=
slope
1
×
2
d
c
The slope and time of the gradient step
in the UHPLC method must be adjusted
so the product of the gradient slope and
dead time remain constant between the
traditional HPLC method and the
UHPLC method.
t
grad
2
2
L
×
L
1
2
F
×
F
2
1
( %
B
)
final
− % B
1
initial
1
=
slope
2
76
Example:
slope
t grad
4.
6
mm
2 150
mm
0.
54
mL/min
= 0. 75% / min×
× ×
5.
83% / min
2
2.1mm 50mm
1.
0mL/min
2
=
( 85% % − 70%
%
)
=
=
2
5.
83% / min
2.
6min
20 min 2.6 min Gradient Step

Transferred Gradient Program
90 HPLC
85
90
UHPLC
85
%ACN
80
75
%ACN
80
75
70
70
65
0 5 10 15 20 25 30 35 40 45
Time(min)
65
0 1 2 3 4 5 6 7 8 9
Time(min)
Comparison of HPLC and the transferred
(calculated) UHPLC gradient programs
Step
HPLC
Time
UHPLC
Time
%ACN
1 0 0 70
2 5 0.6 70
3 25 3.2 85
4 29 3.7 85
5 30 3.8 70
6 45 5.7 70
8X
Decrease in
Predicted Runtime
77
45 min Run 5.7 min Run
UHPLC Applications
34.00
32.00
Nutraceutical ti Application: Ginsenosides id from Ginseng
2
Peak List
1. Rg1
1
2
2. Re
3
3. Rf
30.0000
4. Rb1
4
6
5. Rc
28.00
5
7
1
6. Rb2
26.00
24.00
22.00
7. Rd
3
6
4
20.00
18.00
5
PE Brownlee C 18
7
50 5.0 55 5.5 60 6.0 65 6.5 70 7.0 75 7.5 80 8.0 85 8.5 90 9.0
50 mm x 2.1 mm, 1.9 µm
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5
16.00
14.00
12.00
10.00
PE Brownlee C 18 150 mm x 4.6 mm, 5 µm
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60
78
5X Throughput Improvement

110.00
100.00
90.00
80.00
70.00
UHPLC Applications
Nutraceutical ti Application: Isoflavones in Soy
3
1
4
3
2 5
4
6
Peak List
1. Daidzin
2. Glycitin
3. Genistin
4. Daidzein
5. Gylcitein
6. Genistein
60.0000
50.00
0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5
PE Brownlee C 18
50 mm x 2.1 mm, 1.9 µm
2
40.0000
1
.5 2.0 2.5 3.0
5
6
30.00
20.0000
10.00
PE Brownlee C 18 150 mm x 4.6 mm, 5 µm
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
79
6X Throughput Improvement`
Benefits from Migration to UHPLC
Improve Productivity
• Expected analysis time (t ana) is directly proportional to change in column dead
volume.
1 = HPLC Method
• So:
d
p L
2 2
2 = UHPLC Method
t
ana
= t
2 ana
× ×
1
d p
L
1 1
d p = Particle Diameter
L = Column Length
• 150 mm x 4.6 mm x 5.0 µm 50 mm x 2.1 mm x 1.9 µm
Example:
t ana
1.
9um
50mm
= 45min×
× 5.
7min
5.
0um
150mm
2
=
~ 8X
Throughput Improvement
80
10 Runs/Shift 84 Runs/Shift

Benefits from Migration to UHPLC
Decrease Mobile Phase Consumption
• For successful method migration the product of mobile phase velocity (u) and
particle size (d p ) should be maintained at constant value. [ (u•d p ) 1 =(u•d p ) 2 ]
• The migration of flow rate is dependent on particle size and internal column
diameter.
Example:
F
2
dc
d
2
F
2
= F1
× ×
2
d d
c
1
p
p
1
2
d c = Column Diameter
F = Flow Rate
2
2.
1mm
5.
0um
mL/min × × 0. 54 mL/min
2
4. 6mm
1.
9um
2
= 1 =
2
MP Consumption HPLC = 1.0 mL/min x 45 min = 45 mL
MP Consumption UHPLC = 0.54 mL/min x 5.7 min = 3.1 mL
~93%
Decrease in MP Consumption
Flow Rate
~ 50%
81
Use Less MP and Reduce Waste Disposal
Summary
Migration to UHPLC requires an LC system optimized for UHPLC, not
just a new column
Conversion of HPLC method conditions is simple with the available
UHPLC conversion calculators
Increase productivity up to 9-fold by decreasing runtimes
Decrease consumption of mobile phase by up to 93% and lower waste
disposal cost
Green Technology
Method development/optimization is much faster due to short UHPLC
runtimes
82
Instruments will pay for themselves by increasing productivity and
reducing operating costs.
Don’t Be Afraid of UHPLC

Flexar TM : New HPLC
PerkinElmer Korea
Kim, Wang-Yu PhD
from PerkinElmer
83
© 2009 Perkin Elmer
Flexar. More choices in LC analysis.
18,000 PSI
84
…the most choices in pump pressures

Flexar Features
Bottle tray with
integrated
degassing and
SW comm link
Inter-component
drain
management
Built on rugged
PerkinElmer LC
technology,
recognized for
reliability
Tubing
management for
streamlined
chromatography
Elegant, ergonomic
user interface with
streamlined,
consistent look &
footprint
Controlled by
both Chromera
and TotalChrom
85
…designed with form and function in mind
Flexar Solvent Manager
Three versions available
• Without degassing
• 3-channel degassing
• 5-channel degassing
Can be combined with any Flexar
pumps
Solvent delivery
tubes conveniently
managed through
rear manifold.
Can hold up to five 1–liter
solvent bottles
Built-in communications
link and vacuum degasser
Stackable design, with Flexar
tube management and builtin
inter-component drain
system
Removable tray to contain
breach of complete bottle
86
…more than just a bottle tray

Flexar Autosampler
Easy
Open
Access
87
…absolutely accessible for easy loading and service
Flexar Autosamplers: World-class Performance
Excellent Injection Repeatability
‣ Above 5µL injections, %RSDs of ≤0.5%
‣ For 5µL Linjections, in µL-Pickup mode, %RSDs are also typically ≤0.5%
‣ Area %RSDs fixed loop mode of ≤0.3%
‣ For 2µL injections, in µL-Pickup mode, (10µL loop) area %RSDs are typically ≤0.5%
Anthracene
area precision:
0.24% RSD
5µL injection
Very low
carryover:
Pressure
assist air
purge after
each run
(1) 300 ppm caffeine
n=30
(3) 30µg/L caffeine
(2) Blank injection
88
…what carry over?

Flexar LC Column Oven
Three versions
• Heat only
• Peltier (heat and cool)
• Peltier with column selection/switching
(TotalChrom only – Chromera coming)
Built-in i leak alarm
Integrated solvent pre-heater/chiller minimizes
temperature gradients
• Better column performance
• More repeatable retention times
Temperature range of 30ºC to90ºC (5ºC to
90ºC for Peltier), controlled to within 0.2ºC
throughout entire temperature range
Stackable design, with Flexar
tube management and builtin
inter-component drain
system
Large, easily
accessible column
compartment holds
even 30-cm column
format
89
…Precise temperature control for improved retention time stability.
Flexar PDA Detectors…
FX PDA UHPLC Detector
Excellent performance, HPLC or UHPLC
Improved noise specifications (

Flexar UV/VIS Detectors…
FX UV/VIS UHPLC Detector
World-class UV/VIS detection
Dual beam optical design with
choice of tungsten or deuterium
light sources with wavelength range
of 190-700 nm
Up to 50 pts/sec
10 µL flow cell standard d –
compatible with a wide range of
optional flow cells
Fast, sharp UHPLC performance
High efficiency 2.4 µL flow cell for
optimum UHPLC peak resolution
100 pts/sec detection to capture
even the fastest t UHPLC peaks
UV/VIS LC Detector
91
…sensitive, selective, speed, dynamic range
Flexar Refractive Index and Fluorescence Detectors…
Refractive Index Detector
Combine sensitivity and specificity
Best sensitivity in the market
Now under Chromera control and can
conveniently be combined with
UV/VIS detection (PAH)
92
Rugged general-purpose detection
Highly stable and sensitive LC and
GPC detector for compounds that do
not have high UV absorbance such as
polymers, sugars, organic acids and
triglycerides
Internal temperature of flow cell for
baseline stability
Autozero and autopurge of reference
cell make it easy to use
Fluorescence Detector
…now fully controlled under Chromera!

Flexar UHPLC Systems…
FX-10 UHPLC System
FX-15 UHPLC System
FX-10: High Value UHPLC
Micro Binary 10,000 psi pump package
FX-15: Ultimate in UHPLC
Dual reciprocating i 18,000 psi pump
High efficiency FX UHPLC autosampler
Column oven for retention time
reproducibility and viscosity reduction
High speed FX UV/VIS detector (100
pt/sec) with high efficiency 2.4µL flow cell
93
…You don’t have to pay more to achieve so much.
Flexar FX-15 UHPLC Pump
15,0000 psi operation for the most
demanding UHPLC applications with up to
10x productivity improvement – up to
5mL/min at 18,000 psi!
Green productivity –mobile phase
solvent consumption reduced by 10-15x
Optical Sensor
synchronizes injection
with piston position
Maximum retention time
repeatability for UHPLC
Dual reciprocating 15,000
psi pump
Smoother more precise flow for
retention time repeatability
High pressure Ti-tip check
valves and pulse dampeners
Rated to >15,000 psi operation for
highest throughput UHPLC
Integrated t piston
wash function
Keeps precision pumps
clean even with buffers
94
…the ultimate in UHPLC

Flexar FX-15 Pump for Ultimate UHPLC
Exclusive Ultra High Pressure
Pulse Dampeners
Rated to >18,000 p.s.i.
Significantly reduces pulsation.
Innovative Check “T”
Prevents channel-to-channel cross talk.
No back flow to affect composition.
Improved retention time repeatability.
95
FX-15 UHPLC Pump Piston Drive
Automatic Piston Wash
SAPPHIRE
PISTON
‣ Greatly Increases Seal Life
(particularly with buffers)
MOBILE
PHASE
FLOW OF
FLUSH SOLUTION
PISTON
MOVEMENT
‣ No Auxiliary Pump Required
(self flush)
PRIMARY
HIGH-PRESSURE SEAL
SECONDARY
SELF-FLUSH SEAL
‣ Dedicated recirculation flush
solvent
‣ 15 µL displacement
‣ Self-centering piston saddle
‣ Captive spring for fast head removal
96

Flexar. Reach for more choices in LC analysis.
FX-15
15,000 psi
Autosamplers
FX-10 pump package
FX-15
Pumps
FX-10
10,000 psi
PDA
Flexar
6,000 psi
UV/VIS
RI
Manual injection systems
FL
Ovens
Chromera software
Direct access to
all system
status
parameters
User configurable
Direct access
status panel for
to all
critical parameters
instrument
controls and
settings
Direct access to
any area in the
software
98

Chromera Report
Refine the report style
based upon the needs
of the data
Choose from a
gallery of report
styles
Create and save the specific
layouts needed for
automated reporting
99