HPLC Basics / Instrumentation

Justin Nadar
Application Specialist, HPLC/UHPLC

Proprietary & Confidential

 Understanding the Principles of HPLC

Proprietary & Confidential

 Overview

• Fundamentals of Chromatography – HPLC

• Instrumentation – HPLC

• Applications

3
 INTRODUCTION

4
 History

Mikhail Tswett, Russian Botanist, 1872-1919

In 1906 Tswett used chromatography

to separate plant pigments.

He called the new technique chromatography

Chromatography means color writing

Chroma means “color” and graphein means to “write”

He used liquid –adsorption column chromatography with calcium carbonate as

adsorbent and petrol ether / ethanol mixtures as eluent to separate chlorophylls
& carotenoids

5
 Chromatography

Chromatography is an analytical technique commonly used

for separating a mixture of chemical substances into its
individual components so that the individual components can
be thoroughly analyzed

6
 Chromatography

Column Chromatography

Planar Chromatography

Gas Chromatography

Liquid Chromatography

Super Critical Fluid Chromatography

Simulating Moving Bed Chromatography

7
 Liquid Chromatography

8
 Introduction – The Chromatographic Process

Continous stream of mobile phase

Column Stationary Phase Mobile Phase

• The separation is based on differential partitioning between the mobile

and stationary phases
• Subtle differences in a compound's partition coefficient result in
differential retention on the stationary phase and thus affect the
separation

9
 Introduction – HPLC-System: General Design

• Pump with Degasser

• Autosampler
• Column (installed in a Column Compartment)
• Detector
• Computer with control software

Column
Autosampler

10
 Detectors
Detector Response – Selectivity Sensitivity Linear Range Destructive Flow
Solute Sensitive
4
RI/RID Universal – No microgram 10 No Yes
Bulk
5
UV-PDA Selective - Chromophore nanogram 10 No No
Solute
3
FL/FD Selective - Fluorophore picogram 10 No No
Solute
ELS/ELD/ Selective - Volatization picogram Non-Linear Yes Yes
ELSD Bulk
4
MS Selective- Ionization femtogram 10 - IS* Yes Yes
Solute
5
MS/MS Selective - Ionization femtogram 10 - IS* Yes Yes
Solute attogram
6
ECD Selective - Redox picogram 10 Yes Yes
Solute
4
CAD Selective - Aerosols nanogram 10 yes Yes
Bulk
11
 Chromatography

Characteristics that are to be fulfilled by a detector to be used in HPLC

determination are:

High sensitivity, facilitating trace analysis

Negligible baseline noise to facilitate lower detection

Low drift and noise level

Wide linear dynamic range (this simplifies quantitation)

Low dead volume (minimal peak broadening)

Cell design that eliminates remixing of the separated bands

Insensitivity to changes in type of solvent, flow rate and temperature

Operational simplicity and reliability

Tunability so that detection can be optimized for different compounds

12
 Chromatography

Assay
Related substances
Dissolution
Content Uniformity
Genotoxic impurities
Determination of Molecular weight
Reverse engineering
Characterisation
Carry-over studies
Bioequivalence studies (ADME/PDPK)
Reaction monitoring
Chemical kinetics
Toxicological studies
Clinical and pharmacological studies
13
 Introduction – Characteristics of a Chromatogram

1 - Parabene_Summit_071002 #5 Parabene isocratic 70B Summit 5 UV_VIS_1

2 - Parabene_Summit_071002 #5 Parabene isocratic 70B Summit 5 Pump_Pressure
600 140,0
mAU bar
Retention time

2 - 6,573
1 - 5,363
500 Detector
signal 130,0

3 - 8,863
400
Peak Height
Pressure
120,0

4 - 12,930
signal and
300 ripple

200 110,0

Peak area
100
%D: 0,0 % 100,0
2 %C: 0,0 % Baseline
Methanol: 70,0 %
1
Flow: 0,50 ml/min
min
-50 90,0
0,0 1,0 2,0 3,0 4,0 5,0 6,0 7,0 8,0 9,0 10,0 11,0 12,0 13,0 14,0 15,0

14
 THEORY OF
CHROMATOGRAPHY
Plate Theory and Column Efficiency
Band Broadening (van Deemter plot)
Resolution
Influence of Smaller Particle Diameter

15
 Theory of Chromatography – Plate Theory

• Plate model assumes that the

chromatographic column contains a large
number of separate layers, called
theoretical plates.

Mobile Phase
• These plates do not really exist; they
are a figment of the imagination to help us
describe the processes in the column.
• Within the plates, the mobile and
stationary phases, are described as being
in equilibrium.
Stationary
Phase • The partition coefficient K is based on this
equilibrium and is defined as:
K = cstat / cmob
• As K increases it takes longer for solute
to elute and the retention time increases.

16
 Theory of Chromatography
– Plate Number and Column Efficiency

• The plate theory serves as a way of measuring column quality

• Either by stating the number of theoretical plates (N) in a column (the more
plates the better)

or

• by stating the plate height (HETP, H); the Height Equivalent to a Theoretical
Plate (the smaller the better)

• Columns with high plate numbers are more efficient than columns with
lower plate numbers.

• A column with a high number of plates will have a narrower peak at a

given retention time than a column with a lower number of plates.

17
 Theory of Chromatography
– Influence of Efficiency on the Separation Quality
Formulas are only valid for isocratic methods!
2
 tR  L

N = 5.54⋅   =
wh  wh  H
concentration

2
L  wh 
H= ⋅  
8 ln 2  tR 
H - plate height; N - plate number
L - column length
wh tR - retention time
wh - peak width at half height

Small H or high N means

sharp peaks!
time (length)

N is related to band broadening in the chromatographic system.

18
 Theory of Chromatography
– Causes of Band Broadening

• Extra column effects

• Tubing and connections
• Detector flow cell
• Eluent pre-conditioner
• In-line filter
• In-column effects
• Unwanted processes
• Damaged packing, void volumes
• Immobilized impurities on stationary phase or column inlet frit
• Thermal mismatch
• Natural processes
• Eddy dispersion
• Longitudinal diffusion
• Resistance to mass transfer

Van Deemter Theory explains natural processes.

19
 Theory of Chromatography
– Band Broadening (Van Deemter Plot)

B
H =A + + C ⋅ u
u
A
Plate height (HETP)

u
Resistance to mass
transfer C u
B
Eddy dispersion A
Longitudinal diffusion B/u
C
Mobile phase velocity (u)

20
 Theory of Chromatography
– Parameters which Influence Band Broadening

• Particle diameter
• Column packing quality
• Linear velocity of mobile phase
• Sample characteristic
80
• Eluent viscosity
• Retention factor dp = 10 µm
60 dp = 5 µm
dp = 3 µm
dp = 1.7µm

H [µm]
40

Van Deemter describes 20

only efficiency optimization
but we are actually looking 0
into resolution optimization 0 2 4 6 8 10
u [mm/s]

21
 Theory of Chromatography
Resolution R of two peaks: Goal of every chromatographic
method! • Distance between the peak
centers of two peaks
divided by the average
tR2 base width of the peaks.
• From theory R > 1.50
indicates baseline
Signal

tR1 Peak 2
separation.
• In real life R ≥ 2 is usually
Peak 1
the goal (requested in
regulated environment).
• Much more resolution than
2 does not improve
w1 w2
separation quality but
Time, s
increases analysis time.

22
 Theory of Chromatography

Thermodynamic and Kinetic Factors that determine

Resolution

The fundamental
(α − 1) • k parameters
R = 1/ 4 N •

α k +1 • Efficiency, N
• Selectivity, α
Term for Term for Term for • Retention
Efficiency Selectivity Retention characteristics, k
Characteristics

23
 Theory of Chromatography
– Effects on Resolution

N
α
k
24
 Theory of Chromatography – Selectivity

(tms) t s 2 t ms 2 − t m
α= =
25 t s1 t ms1 − t m
20
Selectivity can be changed by
15
• Changing the stationary phase
10
• Changing the mobile phase or
5
ionic strength
• Changing the temperature
0 (column oven)
0 2 4 minutes 6 8 10 • Changing the charge on the
tm ts solute (pH)
1  α − 1  k 
R= N   
4  α  k +1 

25
 Theory of Chromatography – Retention
Characteristics

(tms) k = ts/tm = (tms-tm)/tm

25

20 • Small k values
• the components elute close to
15 void volume
10 • are not well resolved
• Large k values
5 • good separations
• Very large k values
0
• longer retention times
0 2 4 minutes 6 8 10
• peak broadening which can
tm ts compromise sensitivity
1  α − 1  k  • Typical working range
R= N   
4  α  k +1 
• 2 < k < 10

26
 Theory of Chromatography
– The Influence of the Particle Diameter on Speed

250 x 4.6 mm; 5 µm

100
L/dp = 50
10 µm particles 1.5 mL/min
5 µm particles
3 µm particles 150 x 4.6 mm; 3 µm
Speed 2.8x
H [µm]

2 µm particles
L/dp = 50
No loss in
uopt, 3 µm 2.5 mL/min resolution
uopt, 5 µm uopt, 2 µm
Hmin, 5 µm
Hmin, 3 µm 100 x 4.6 mm; 2 µm
Hmin, 2 µm Speed 6.2x
0 L/dp = 50
0 5 10 No loss in
Linear Velocity u [mm/s] 3.75 mL/min resolution min
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0

27
 Theory of Chromatography
– Influence of the Particle Diameter on Pressure

Efficiency Pressure
100 1200

1000
2 µm particles 2 µm particles

Column pressure [bar]

3 µm particles 3 µm particles
5 µm particles 800 5 µm particles
10 µm particles 10 µm particles
H [µm]

600

400

200

0 0
0 2 4 6 8 10 0 2 4 6 8 10
Linear Velocity u [mm/s] Linear Velocity u [mm/s]

For separation speed – We Need High Pressure and Fast Flows

28
 Introduction – Size of Silica Particles

• The smaller the particles, the better the separation performance

• But: Smaller particles generate higher back pressure

Typical Typical Typical

Particle Size Back Pressure Column Diameter
Preparative HPLC 100 – 10 µm 10 - 100 bar 21 mm
Conventional HPLC 5 – 3 µm 100 – 300 bar 4.6 mm
UHPLC ≤ 3 µm ≥ 600 bar 2.1 mm

• HPLC: High Performance Liquid Chromatography

• UHPLC: Ultra High Performance Liquid Chromatography

29
 Theory of Chromatography – Summary

• Plate theory and column Efficiency

• The higher the plate number N the better the efficiency of the column

• Band broadening (van Deemter plot)

• Natural processes result in band broadening

• Resolution is a function of column efficiency, selectivity and retention

characteristics.

• Influence of smaller particle diameter:

• Higher resolution
• More speed possible
• Higher back pressure generated

30
 Normal Phase Chromatography
Reversed Phase Chromatography
Isocratic elution
Gradient Elution
Mobile Phase Additives

COLUMN CHEMISTRY

31
 Column Chemistry – The Column

• Solid Support - Backbone for bonded phases

- Usually porous silica or polymeric particles

• Bonded Phases
- Functional groups chemically bound to the solid support.

• Frits or Sieves as Stationary Phase Retainers

- Protects the packing from sample matrix particles
- Mesh size shrinks with particle size  ≤ 3 µm require thorough eluent
filtration!

32
 Column Chemistry – HPLC-Modes

• Normal Phase (NP): The stationary phase is polar and the mobile
phase is non-polar.

Niche mode in modern (prep) LC (e.g. separation of enantiomers)

• Reversed Phase (RP): The stationary phase is made of chemically

modified silica (normally with non-polar surface) and the mobile phase
is polar.

Most common HPLC mode

Good for separation of broad polarity range

• Early HPLC experiments worked with pure silica and polar solvents.

General rule: ”Similia similibus attrahuntur“ (“like dissolves like”).

33
 Column Chemistry
– Reversed Phase Chromatography

Example: C8

Si • Common RP stationary phases:

O Normal silica treated with RMe2SiCl
Si R=(CH2)17CH3: C18
O R=(CH2)7CH3: C8
Si
O • Common RP mobile phases:
Si Water < Methanol < Acetonitrile
(elutropic series for RP: decreasing retention)
Silica- C8

34
 Column Chemistry
– Mobile Phase Additives in Reversed Phase

• Inorganic salts:
• Altered surface tension affects retention characteristics by altering the
analyte/surface interaction
• Example: Ammonium sulfate for peptide separation
• Ion pairing reagents
• Neutralize charged analytes for better retention
• Example: Trifluoroacetic acid (TFA), Triethylamine (TEA)
• Buffering agents
• Neutralize charged analytes for better retention and increase reproducibility
of retention time
• Example: Sodium acetate buffer, sodium phosphate buffer

35
 Column Chemistry
– Separation of Acidic, Basic and Neutral Compounds
90
mAU WVL:254 nm

2 Aniline
75
C18, 2x100 mm, 1.8 µm
63 H2O/MeOH (50/50), 450 µL/min
1 - Uracil 4 - p-Ethylaniline

50
3-
Phenol
38 5 Toluene

25
6 - Ethylbenzene

13

min
-10
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0

Basic compound: Acidic compound: Neutral compound:

Aniline pKb=2.7 Phenol pKa=9.9 Toluene

36
Unpolar compounds have longer retention times
 Column Chemistry – Acids and Bases – pH Effects

Acids Base

Practical HPLC Method Development

Snyder, Glajch, Kirkland 1988

The stronger an analyte is charged, the less its retention on a RP-Phase

37
 Eluent
Capillaries
Column
Pump
Autosampler
Oven
Detector

SYSTEM CONTRIBUTION

38
 System Contribution –
How do the Modules Contribute to the Chromatogram?

Pump Retention time

Pressure ripple
Detector baseline noise

Autosampler Peak area

Oven Retention time

Sensitivity

Detector Baseline noise

Sensitivity

39
 System Contribution – Summary

• Eluents have to be prepared and handled with reasonable care to

avoid particle contamination.
• Dead-volume free connections and minimized extra-column volume by
use of appropriate tubing and fitting systems.
• The pump has to mix the eluents precisely and accurately and provide
a constant flow rate and back pressure.
• The autosampler has to work accurately for successful method
transfer and preciscely for good result reproducibility.
• The temperature effect compound retention and peak shape and has to
be controlled reliably by the column oven.
• Reasonable detector settings are essential for good peak mapping.

40
 Dionex’ UHPLC-Compatible Detectors

VWD MWD DAD

100Hz VWD-3100 100Hz MWD-3000SD 100Hz DAD-3000SD

200Hz VWD-3400RS 200Hz MWD-3000RS 200Hz DAD-3000RS

FLD ECD Corona Ultra (CAD)

100Hz FLD-3100
200Hz ECD-3000RS 200Hz Corona Ultra RS (CAD)
200Hz FLD-3400RS

The most complete UHPLC detector range

Note: 200 Hz only available with Chromeleon 7

41 Proprietary & Confidential

 M16

Vanquish HPLC / UHPLC System

• Integrated modularity
• Biocompatible, iron-free flow path (default)
• Viper-based, tool-free fluidic connections
• Support of latest innovation in column technology
• Enhanced LC/MS support and integration
• Portable system control with tablet PC (planned)
• Revolutionary module drawer system
for outstanding service accessibility
• Removable doors for easy access
• Reduced system height
• Variable system GDV in standard configuration as
low as 40 µL

42
Slide 42

M16 Slide 7 bis 12 kommen auch in der Customer-Präsentation vor - da sind sie auch verfeinert worden
MNeubaue, 6/30/2014
 Universal UHPLC Detection with Corona Ultra

UV
5 Peaks

Corona
9 Peaks

Separation of 9 pharmaceutical compounds in less than 5

minutes

43
Vitamin D - Coulochem III and ultra Analytical cell

1 – Internal Std
2 – D3
325
3 – D2
1
300
2
Current / nA

3
275 500 ng on column
Run Time = 3 min
250

225

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Time / Min.

44
 AN109: Rapid HPLC Separation of Multiclass Antibiotics
in Food and Water

F&B

Column: Dionex Acclaim RSLC PA2, 2.2 µm, 150 x 2.1 mm

45
 Separation of Fat-Soluble Vitamins on Acclaim RSLC PolarAdvantage II
(PA2)

F&B

Column: Acclaim RSLC PA2, 2.2 µm, 100 x 2.1 mm

46
 AN245: Fast HPLC Analysis of Dyes in Foods and Beverages

F&B

Column: Dionex Acclaim RSLC PA2, 2.2 µm, 50 x 2.1 mm

47
 Anthocyanidins analysis with Standard System
70,0

2 - Cyanidin
Billberry extract Analysis Time: 2 min
Max Pressure: 600 bar

1 - Delphinidn
Column: AcQuity BEH, C18,1.7 µm

4 - Pelargonidin
mAU
Dimensions: 50 x 2.1 mm ID
Eluent: A: 0.3% H3PO4
B: ACN
Temperature: 40 oC
Flow rate: 1.0 mL/min
-5,0 Injection Vol: 1 µL sample in 80% EtOH
80,0 Detection: UV 520 nm
Pomegranate Juice Peaks: 1. Delphinidin 2. Cyanidin
1 - Delphinidn

3. Petunidin 4. Pelargonidin
2 - Cyanidin

5. Peonidin 6. Malvidin
3 - Petunidin

6 - Malvidin

Gradient Table: 0.00 min 10.0% B

5 - Peonidin

mAU
2.00 min 20.0% B

-5,0
0,00 0,50 1,00 1,50 2,00 min

48
 Ultrafast Analysis of Water Soluble Vitamins
by Parallel UHPLC

F&B

Column: Dionex Acclaim RSLC PA2, 2.2 µm, 100 x 2.1 mm

49
 AN193: Determination of Additives in Carbonated Beverages using
Mixed-mode WAX-1 columns

F&B

Column: Acclaim Mixed-Mode WAX-1, 5 µm, 150 x 4.6 mm

50
 High-Resolution Triglyceride Profile of Cooking Oils

F&B

Column: Acclaim RSLC 120 C18, 2.2 µm, 100 x 2.1 mm

51
 EPA-PAHs in Tea Samples with Standard System

fluoranthene
10.0

Benzo(k)-
A: EPA-PAH Standard, 10
ng.mL-1 Column: MN Nucleodur C18

Benzo(b)fluoranthene
Phenanthrene
Anthracene

Benzo(a)pyrene

Indeno(1,2,3-cd)pyrene
PAH,

Benzo(g,h,i)perylene
B: Black Tea, St. Petersburg

Pyrene
C: Black Tea, London 3 µm (100 x 3.0 mm)
5.0 D: Korean Green Tea
Eluents: A:Water

Fluoranthene

Chrysene
E: Brazilian Mate Tea
B:Acetonitrile
Gradient: 0 – 0.5 min: 45% B
107 cts

A 45 – 90% B in 3.262
0 min
B 90 – 95% B in 0.513
min
C 95% B for 1.013 min
Flow Rate: 2.0 mL/min @ 537 bar
D
Temperature: 30 °C
E Inj. Vol.: 12.5 µL
Detection: FLU
Sample: see fig.
0 1.0 2.0 3.0 4.0 5.0 6.0
Minutes

52
 Polyphenols Analysis with RSLC System

53
 Carotenoides Analysis with RSLC System

54
 AppsLibrary Conclusion
• Web Based Application Search Engine

• Offers easy search option for Dionex IC/LC applications

• Provides unique structured searching tools for

• Analyte name or class
• Market and instrument type
• Column information such as particle size and packing
material

• Modern application view screen

• See all details in a single glance

• Unique download of all application settings for immediate

use

55
 Thank You!

Proprietary & Confidential © 2012 Thermo Fisher Scientific

 QUIZ
• NAME THE LAW THAT FORMS THE PRINCIPLE OF UV DETECTORS.
• NAME THE LAW THAT FORMS THE PRINCIPLE OF REFRACTIVE INDEX
DETECTOR.
• NAME THE EQUATION THAT DEFINES THE PEAK BROADENING WITHIN
A CHROMATOGRAPHIC COLUMN.
• IN REVERSE PHASE CHROMATOGRAPHY, THE STATIONARY PHASE IS
_______________________. (POLAR/NON-POLAR)
• IN NORMAL PHASE CHROMATOGRAPHY, THE MOBILE PHASE IS
________________________ (POLAR/NON-POLAR)
• WHAT DOES THE ‘N’ STAND FOR IN RESOLUTION EQUATION?
• WHAT DOES THE ‘α’ STAND FOR IN RESOLUTION EQUATION?
• WHAT DOES THE ‘k’ STAND FOR IN RESOLUTION EQUATION?
• IN FLUORESCENCE DETECTION, THERE ARE TWO WAVELENGTHS,
WHAT ARE THEY?
• FOR ____________________ DETECTION, THE COMPOUND SHOULD
HAVE REDOX PROPERTIES.

57