Detection in HPLC

Detection Qualification and Types

of Detectors in HPLC
Dr. Shulamit Levin
Medtechnica
http://www.forumsci.co.il/HPLC

Dr. Shulamit Levin, Medtechnica

Detection in HPLC

Detectors
The most common HPLC detectors:
UV/Vis
Fixed wavelength
Variable wavelength
Diode array
Refractive index
Fluorescence
Electrochemical

Beer's Law
Absorbance = Extinction Coefficient x
Pathlength x Concentration
Only for monochromatic light

Less common:
Conductivity
Mass-spectrometric (LC/MS)
Evaporative light scattering

Reduce Pathlength

Single Wavelength UV Detector

Beer's Law

Optical Light Path

Absorbance = Extinction Coefficient x Pathlength x Concentration

Reduce Concentration

Wavelength
Aperture Plate

B
Mercury, Zinc
or Cadmium
Source Lamps

Wavelength
Filter

Reference side

Absorbance AU

Transmittance %

Extinction Coefficient
T=solvent/sample

*
*
*
*
Concentration

Dr. Shulamit Levin, Medtechnica

A=
log(solvent/sample)

*
*
*
*
Concentration

AU
0
1.0
2.0
3.0

%T
100
10
1
0.1

T
1.000
0.100
0.010
0.001

Sample side
Flow Cell

Dual
Photodiode

Detection in HPLC

UV Detection of AccQ-Tag Amino

Acid Derivatives

UV-VIS Detector Optical Bench

SampleName: Cult Std Vial: 1 Inj: 1 Ch: 486 Type: Standard

Optical Light Path

Taper-Cell
Flow
Cell

0.024

Tyrosine
Cysteic Acid
Vaine
Mtehionine

Lysine

Proline
Alpha-aminobutyric acid

Glycine
Glutamine

0.002

Tryptophan

Rotating
Diffraction
Grating
190 to 600nm

Phenylalanine

0.004

Deuterium
Arc Lamp

Isoleucine
Ornithine
Leucine

0.006

Serine

0.008

Aperture
Slit

Glutamic Acid

Illumination
Lens

0.010

Hydroxyproline

0.012
Beam-Defining
Apparatus

Asparagine

0.016
AU 0.014
Aspartic Acid

Sample side

0.018

NH3

AMQ

0.020
Dual
Photodiode

Reference side

Histidine
Arginine
Threonine
Alanine

0.022

Beam Splitter
Mirrors

0.000
15.00

20.00

25.00

30.00

35.00
Minutes

40.00

45.00

50.00

55.00

Extraction of 3D Data

Absorbance

Absorbance

Chromatogram

Liquid from
column

Dr. Shulamit Levin, Medtechnica

1
2

Spectrum
Time

Wavelength

Detection in HPLC

PDA Spectrum Index Plot

Maximum Impurity Detection

DNPH Derivatives 0.25 ng Each Peak

Millennium PDA Spectrum Index Plot - SampleWeight 0.25 ng - PDA 360.0 nm

nm

Millennium PDA Spectrum Index Plot - SampleWeight 0.25 ng 360nm 996PDA 360.0 nm

440.00

440.00

420.00

420.00

440.00

440.00

400.00
380.00

400.00

360.00

360.00

340.00

340.00

320.00

320.00

300.00
280.00

300.00
280.00

260.00

260.00

420.00
400.00
380.00
360.00
340.00
320.00
300.00
280.00

420.00
400.00
380.00
360.00
340.00
320.00
300.00
280.00

260.00

260.00

380.00

0.0006
0.0004
AU

nm

nm

0.0006

0.00030

0.0004
AU

0.00020

0.00030
0.00020

AU

AU
0.00010

0.0002

0.0002

Coelution of DNPH
Hexaldehyde and
2,5-Dimethylbenzaldehyde
Hexaldehyde

0.00010

2,5-Dimethylbenzaldehyde

0.00000

0.00000

0.0000

0.0000

-0.00010

0.00010
18.40

4.00

6.00

8.00

10.00

12.00
Minutes

14.00

16.00

18.60

18.80
Minutes

18.00

PDA and fluorescent Detector

Comparisons for Aflatoxin Analysis

19.00

19.20

Photo-diode array
Chromatographic and Spectral Sensitivity

SampleName: Aflatoxin Mix Vial: 2 Inj: 1 Ch: SATIN Type: Standard

55.00

45.00
40.00
35.00
mV 30.00

Aflatoxin
B1

25.00
20.00
15.00

1.6

10.00

2.4

Minutes

5.00
0.00
4.00

5.00

Dr. Shulamit Levin, Medtechnica

6.00

7.00
8.00
Minutes

0.07mAU
0.530 AU

0.07 mAU
0.15 ng Ethylparaben

UV at 360 nm 31 point smoothed

UV at 360 nm
Fluorescence 365 ex 455 em
Aflatoxin
B2
Aflatoxin
G1
Aflatoxin
G2

Absorbance

50.00

9.00

10.00

11.00

12.00

nm

3.2

210.0

250.0

nm

290.0

Detection in HPLC

Detectors
The most common HPLC detectors:
UV/Vis
Fixed wavelength
Variable wavelength
Diode array
Refractive index
Fluorescence
Electrochemical

Differential Refractive Index Detector

No sample = n

S
R
LAMP

With sample = n+

LED or
Incandescent

Less common:

Conductivity
Mass-spectrometric (LC/MS)
Evaporative light scattering

To Amplifier

R
X = Const x n

Refractive Index Detection with

Differential RI - Sugars

Refractive Index Detection with

Differential RI - Sugars

SampleName: Sugars D Vial: 1 Inj: 1 Ch: SATIN Type: Standard

120.00

SampleName: Sugar Stds -500 ng each

Fructose

100.00

Dextrose

Sucrose

80.00

0.0000004

Sucrose

60.00
Maltose

40.00

Lactose

20.00
0.00
mV

Dextrose
Fructose

del
RIU

-20.00

0.0000002

-40.00
-60.00
-80.00
-100.00
-120.00

00000000

-140.00
-160.00

Bagel Extract
5.00

6.00

Dr. Shulamit Levin, Medtechnica

7.00

8.00
Minutes

5.00
9.00

10.00

11.00

5.50

6.00

6.50

7.00
Minutes

7.50

8.00

8.50

9.00

Detection in HPLC

Refractive Index Detection with

Differential RI - Polymers

Sensitivity
Refractive Index Detector

10300

800.00
750.00
700.00
96400

190000

Styragel HR 0.5,
4.6 x 300 mm,
35C, 0.35 mL/min
dRI sensitivity =
32X, 32C

Del RIU

300.00
250.00
200.00
150.00

192300
Dow 1683

5.0
18.00

20.00

22.00

24.00
Minutes

26.00

28.00

Less common:
Conductivity
Mass-spectrometric (LC/MS)
Evaporative light scattering

7.0

8.0

Fluorescence Detectors

The most common HPLC detectors:

UV/Vis
Fixed wavelength
Variable wavelength
Diode array
Refractive index
Fluorescence
Electrochemical

6.0

Minutes

30.00

Detectors

Dr. Shulamit Levin, Medtechnica

1=Tristearin
2=Myristic acid

1260000

2890000

450.00
400.00
350.00

100.00
50.00
0.00

250 ng on column
1

650.00
600.00
550.00
500.00
MV

5570

SampleName: GPC STDS

Excitation filter
Cell

LAMP

Emission filter

Photomultiplier
Short pass - transmits all wavelengths below a specified cutoff
Long pass - transmits all wavelengths above a specified cutoff
Band pass - blocks all wavelengths outside a specified band

Detection in HPLC

Sensitivity
Fluorescence Detector Optical Bench

Emission
Grating

Photomultiplier
tube
Mirror

Emission
Slit

Flow
Cell

Torroidal Mirror

Excitation
Grating

Fluorescence Detector

Beam Splittter
Excitation
Slit

Photo
diode

0.1 pg Anthracene
Excitation = 251 nm
Emission = 406 nm

mV

5 mV
Torroidal Mirror
LAMP

Mirror

0.0

1.0

2.0

3.0

4.0

5.0

Minutes

Fluorescence vs. UV Detection

The most common HPLC detectors:

AMQ

AccQ-Tag amino acid

analysis

Response

Fluorescence
Excitation=250 nm
Emission=395 nm
UV 254 nm

20.00

40.00

Minutes

Dr. Shulamit Levin, Medtechnica

Detectors

60.00

UV/Vis
Fixed wavelength
Variable wavelength
Diode array
Refractive index
Fluorescence
Electrochemical

Less common:
Conductivity
Mass-spectrometric (LC/MS)
Evaporative light scattering

Detection in HPLC

Electrochemical Detection of
Catecholamines & Related Compounds

Electrochemical Detector
Reference Electrode

Working Electrode

Analyte is oxidized or reduced

1.
2.
3.
4.
5.
6.
7.

Norepinepherine
Epinepherine
Normetanepherine
Dopamine
Metanepherine
3-Methoxytyramine
4-Methoxytyramine

nAmps

150 ppb
200 ppb
50 ppb
200 ppb
200 ppb
75 ppb
500 ppb

5
7
6

Electrolyte (mobile phase)

Auxiliary Electrode
As compounds are oxidized or reduced, a current proportional to concentration is produced.

0.00

2.00

4.00

6.00

8.00

10.00

12.00

Minutes

Pulsed Amperometric Detection

of Monosaccharides
1.
2.
3.
4.
5.
6.

300

4
6

Less common:

0.00
5.00

Dr. Shulamit Levin, Medtechnica

Minutes

The most common HPLC detectors:

UV/Vis
Fixed wavelength
Variable wavelength
Diode array
Refractive index
Fluorescence
Electrochemical

Fucose
Galactosamine
Glucosamine
Galactose
Glucose
Mannose

mV

Detectors

20.00

Conductivity
Mass-spectrometric (LC/MS)
Evaporative light scattering

Detection in HPLC

Conductivity Detection of Seven

Anion Standard

Conductivity Detector

1.
2.
3.
4.
5.
6.
7.

1.40
3
2

Mobile phase

Fluoride
Chloride
Nitrite
Bromide
Nitrate
Phosphate
Sulfate

1 ppm
2 ppm
4 ppm
4 ppm
4 ppm
6 ppm
4 ppm

Column:
Eluent:
Flow rate:
Injection vol.:
Detection:

1.05

5
7

Mobile phase plus sample

0.70
0.00

5.00

Conductivity and UV Detectors in

Series
1.60

Detection:

Direct Conductivity after

Suppression

1.20
4

0.80
S

7
6

0.40
0.00
3

0.05

Fluoride
Chloride
Nitrite
Bromide
Nitrate
Phosphate
Sulfate

0.03
AU
0.02

Flow rate:
Injection vol.:

0.01

4
12.00
16.00
Minutes

Dr. Shulamit Levin, Medtechnica

Minutes

20.00

24.00

15.00

Waters IC-Pak Anion HR

1.2 mM Sodium Carbonate/
1.2 mM Sodium Bicarbonate
1.0 mL/min
50 L

20.00

25.00

Applications
Sensitivities for compounds such as phenol, catecholamines,
nitrosamines, and organic acids are in the picomole (nanogram)
range.
The mobile phase must be made electrically conductive, usually
by the addition of a suitable salt:

Reversed Phase and Ion-Pair RP

No normal phase separations

0.00
8.00

10.00

Ion Exchange
Column:
Eluent:

4.00

1 ppm
2 ppm
4 ppm
4 ppm
4 ppm
6 ppm
4 ppm

Detection: UV (PDA) at 214 nm

0.04

0.00

1.
2.
3.
4.
5.
6.
7.

Waters IC-Pak Anion HC

Borate/Gluconate
2.0 mL/min
100 L
Direct Conductivity

Detection in HPLC

EVAPORATIVE LIGHT SCATTERING

Detectors
The most common HPLC detectors:
UV/Vis
Fixed wavelength
Variable wavelength
Diode array
Refractive index
Fluorescence
Electrochemical

The scattered light is detected by a

silicone photodiode located at a
90 angle from the laser. The
photodiode produces a signal
which is sent to the analog
outputs for collection. A light trap
is located 180 from the laser to
collect any light not scattered by
particles in the aerosol stream.

Less common:
Conductivity
Mass-spectrometric (LC/MS)
Evaporative light scattering

How LC-MS Works

Radioactive Detector
Primarily used for the measurement of 3H, 14C, and 32P,
beta-emitters and many soft gamma and positron emitters
encountered in bio-medical research and pharmaceutical quality
control.

Ionization

Source

Sorting of Ions

Detection

Ion
Detector

Analyzer

Data
System

LC/MS
Interface
Desolvation

HPLC

Separation

Dr. Shulamit Levin, Medtechnica

Date
Processing

Mass
Spectrum

Detection in HPLC

TTiim
M
A
mee O
Off FFlliigghhtt M
Maaassssss A
Annnaaalllyyyzzzeeerrrsss

FT--ICR
FT
ICR--Spectrometer

SSOOUURRCCEE

LC-MS

R
RE
EF
F LL E
EC
C TT R
RO
ON
NO
OFFFF

D R II FFTT TTUUBBEE

Magnetic Fielt B

DDEETT E
ORR
EC
C TT O
LLI INNEE A R MMO
OD
DE

D
DE
ET
TE
EC
CT
TO
OR
R
R
RE
EF
FL
LE
EC
CT
TR
RO
ON
N M
MO
OD
DE
E

Y
Z

Source

Sender

R
RE
EF
F LL EECCTTRROONN OONN

SSOOUURRCCEE

Trapping Plates
Electrodes

Elektroden

D
RR
DE
E TTEECCTTOO
L
L II N
NEEAARR M
MO
ODDEE

DRIFT
D
R I F T TUBE
TUBE

DC

D
DE
ET
TE
EC
CT
TO
OR
R
RE
EF
FL
LE
EC
CTTRRO
ONN M
R
MO
OD
DE
E

Receiver Plates

Filament
DC

Total-Ion-Current Chromatogram
with poor resolution

Transmitter Plates

1: ScanES+
4.34e5

262.87

100

Ion Traps

DC

Transferoptic

Mix
(10.696)

199

Types of
Mass
Spectrometers
Analyzers

59.99

213.90
%

222.87
235.87

Mixture

263.87

1: Mass Chromatogram
195.98

4.65 5.05

Int.

240.88

264.85

120.80

68.92

98.85

267.91

128.82

76.87

8.62

287.01

309.02

170.92

333.84

0
60

80

100

120

140

160

180

200

220

240

260

280

300

320

m/z

End Cap Electrode

Axial Modulation

+ + +
++ +
++

Ring Electrode, R f

Inlet

ElectronMultiplier

340

190

The Quadrupole
Quadrupole Analysator

5.65

S e c t o r Mass
Sector
Mass Spectrometers
Spectrometers

8.02
Nier -J o h n s o n -G e o m e t r y ( E B )

resonant Ion

Slit

non resonant Ion

10.62

Magnetic sector

Detector

Electrostatic Sector

3.82

(ESA)

0.74
Detector

Slit
dcand Rf Voltages

Ion
Source
IonSource

77

Time
2.00

4.00

6.00

8.00

10.00

Fast LC-MS Analysis

2.1 x 50 mm ( 5 m)
2.56

100

MW=295
8.11e4

0
MW=280
2.21e5

0
MW=264
1.26e5

(4)

1.29

MW=260
1.53e5

Ding

280 1.13e5

100

(2)

281

100

264

(3)

1.57
2.16
1.29
2.56

1.00

2.00

TIC
TIC
3.50e5

3.00

Time (min)

Dr. Shulamit Levin, Medtechnica

4.00

5.00

7.67e4

265

NH

0
100

O
OH

100

296

233

0
100

100

2.16

100

295 4.59e4

1.57

100

HARDWARE
HARDWARE -- ES/APCI
ES/APCI Ion
Ion Source
Source
N

N
H

(1)

260

9.25e4

261

0
100 125 150 175 200 225 250 275 300
Mass/Charge (m/z)
10 L injection of 200 ng/mL sample (in 40%
MeOH),1=Propranolol, 2=Doxepin, 3=Nortriptyline,
4=Trimipramine, 65/35 0.1 % Formic Acid / MeCN
0.2 mL/min

ESI and APCI are easily interchangeable in seconds without

venting the system
ESI and APCI use a unique counter electrode to optimize
sampling from the liquid spray and to aid sample desolvation
Automatic Probe recognition

220

Detection in HPLC

ESI-MS
ESI-MS Ion Formation

Mass Range
Multiply Charged Molecules

Horse Heart Myoglobin

n = 23, m/z = 738
n = 22
n = 21
n = 20
n = 19
n = 18
n = 17
n = 16, m/z = 1060

Acquired Mass range

Liquid Chromatography- Mass Spectrometery

(LC-MS)

APCI
APCI Probe
Probe Equipped With a
Heated
Heated Nebulizer
Nebulizer

Makeup Gas

Heater Block
From LC
Column

SMSSM
S
SSSMM
S

Nebulizer
Gas

+
+ SH M
SH

SH+ +
+
SH M
SH+

+
MH +
S

760 torr

To Mass
Analyzer

vacuum

Corona Discharge
Needle

Generates molecular weight and structural information

APCI flow rate: 0.2 to 2mL/minute
Option: Crossflow interface

Dr. Shulamit Levin, Medtechnica

Calculated Mass

Detection in HPLC

D
D aa uu gg hhhttteeerrr (( P
P rr oo dd uu cc ttt))) II oo nn S
S pp ee cc tttrrraaa
M S 11

C o l l ii ss iioonn
CCee ll ll

S t aatti icc

M
M SS22

Collision
Collision
CCell
ell

MS2

Multiple Reaction
Reaction
Monitoring
Reaction Monitoring
Monitoring

Typically
Typically used
used in
in
in Quantitative
Quantitative Work
Work
Work of
of
of
Triple Quadrupoles
Quadrupoles
Quadrupoles
Quadrupoles
MS1
MS1

S
Sc
c aa n
nnniinngg

Collision
Collision
Cell
Cell

MS2
MS2

Static
Static

C
S pp ee cc tt rr aa
Coo nn ss tt aa nn tt N e u t r a l L o s s S
M
S 11
MS

BASIC DETECTOR REQUIREMENTS

An ideal LC detector should have the following properties:

SSccaa nn nn ii n g

P a r e n t ( P r e ccuurrssoorr)) IIoonn SSppeeccttrraa

M S 11

Triple Quadrupoles
Quadrupoles MSMSMS
Modes of Operation

CCoolllliissiioonn
CCee ll ll

S
Sc
c aa n
n nn iinngg

Static
Static

Static
Static

M
MS
S2
2

S
c aa n
n nn ii nngg
Sc

Low drift and noise level (trace analysis).

High sensitivity.
Fast response for high performance systems.
Wide linear dynamic range (quantitation).
Low dead volume (minimal peak broadening & remixing of the
separated bands).
Insensitivity to changes in type of solvent, flow rate, and
temperature.
Operational simplicity and reliability.
Tuneable, so that detection can be optimized for different
compounds.
Preferably non-destructive.

PROPERTIES OF DETECTORS
Detector Criteria
Selectivity
Sensitivity and detection limit
Stability
Linear range
Dynamic Range
Reproducibility
Effect on peak shape
Maintenance

Dr. Shulamit Levin, Medtechnica

SELECTIVITY

SPECIFIC

UNIVERSAL

A selective detector allows one to see

only components of interest despite of
their co-elution with any others.

Detection in HPLC

PROPERTIES OF DETECTORS
SENSITIVITY
R
E
S
P
O
N
S

PROPERTIES OF DETECTORS
DETECTION LIMIT

h signal = 2 x h noise

h signal

E
CONCENTRATION

Sensitivity of a detector is not the minimum

amount that can be detected.

Detector Sensitivity

h noise

Chromatographic Sensitivity
Signal-to-Noise Ratio

Limit of detection
Lowest concentration that can be detected
Signal-to-noise ratio of 2:1 or 3:1
0.001
AU

0.2 AU

Limit of quantitation
No
apparent
noise

Lowest concentration that can be determined

with acceptable precision
Signal-to-noise ratio of 10:1
2.8

3.0

3.2

Minutes

Dr. Shulamit Levin, Medtechnica

3.4

Noise

2.00

3.00
Minutes

4.00

Detection in HPLC

Factors Increasing UV Signal

Increase Signal-to-Noise Ratio
6:1

3:1

Signal-to-noise (S/N)
is peak height to
noise

8:1

Increase S/N by
increasing peak
height
Increase S/N by
decreasing noise

Increase sample concentration

Increase injection volume
Choice of wavelength (s)
Low volume flow cell
Flow cell pathlength

Factors Affecting Noise in UV Detectors

Optics bench design
Lamp energy
Wavelengths
Mobile phase composition
Pump pulsation
Electronics

Chromatographic Sensitivity

PROPERTIES OF DETECTORS

Single Wavelength vs Maxplo t

BASELINE STABILITY

0.010

Maxplot

220 nm

0.008

SHORT RANGE

AU

0.006

NOISE

0.004
0.002
0.000

LONG RANGE

-0.002
0.0

2.0

4.0

Minutes

Dr. Shulamit Levin, Medtechnica

6.0

0.0

2.0

4.0

Minutes

6.0

DRIFT

Detection in HPLC

Noise and drift

PROPERTIES OF DETECTORS
LINEAR RANGE

Noise, drift, and smallest detectable peak.

R
E
S
P
O
N
S
E

Max linear response

2 x detector noise

CONCENTRATION

The linear dynamic range of a detector is the maximum

linear response divided by the detector noise.

PROPERTIES OF DETECTORS
DYNAMIC RANGE

R
E
S
P
O
N
S
E

CONTRIBUTION TO BAND
BROADENING

RESPONSE TIME

TIME (MIN)

Dr. Shulamit Levin, Medtechnica

PROPERTIES OF DETECTORS

FLOW-CELL VOLUME

Detection in HPLC

PROPERTIES OF DETECTORS
REPEATABILITY OF RESPONSE

PROPERTIES OF DETECTORS
MAINTENANCE AND COST

EASY HANDLING OF FLOW-CELL

EASY A/D CONVERSION
TEMPERATURE, FLOW RATE, ELECTRONICS

SAFETY

Dr. Shulamit Levin, Medtechnica