HPLC can be used better

Great tips for better

results!
Shimadzu Corporation
Analytical & Measuring Instruments Division
JAIMA SHOW 2003 New Technology Briefing
September 11, 2003 Makuhari
Messe

For better analysis in HPLC

Mobile phase
Buffer solution / solvent selection, gradient optimization, flow rate selection
Degassing, flow rate stability, mobile phase preparation

Column
High separation, secondary interaction, lot variation, temperature control

Detection
Sensitivity, selectivity, ambient temperature fluctuation affect

Sample
Extraction method selection, sample solvent selection, dissolved oxygen

Instrument
Performance, stability, durability, ease-of-use

Evaluation overall
Stability of analytical technique, robustness, quantitative accuracy

 Mobile Phase

Solvent Mixing Ratio Notation and Preparation (1)

Is the composition of the following solutions the same?
water / ethanol = 1/1 (v/v)
50%(v/v) ethanol aqueous solution

* Beware of volume change when mixing organic solvents!

The density of the solvent mixture is not a simple
average of the original solvent densities.
If 50mL of water and 50mL of ethanol are mixed, the
final volume becomes about 96mL at about 25C.

 Mobile Phase

Solvent Mixing Ratio Notation and Preparation (2)

water/ethanol=1/1 (v/v)
Measure 500mL each of
water and ethanol, and
then mix
The total is not 1L.

Water

Ethanol

50%(v/v) ethanol aqueous sol.

Transfer 500mL ethanol to 1L
volumetric flask, bring to volume
with water.
The water percentage is high.

Ethanol

1L

Water

 Mobile Phase

Solvent Mixing Ratio Notation and Preparation (3)

Mobile phase A: 20mM phosphoric acid (Na) buffer solution <pH 2.5> /
acetonitrile = 9 / 1 (v/v)
Mobile phase B : 10%(v/v) acetonitrile in 20mM phosphoric acid buffer
solution <pH 2.5>
Peaks
1: acetaminophen
2: dihydrocodeine
3: caffeine

Column

:Shim-Pack VP-ODS
(150mmL. x 4.6mmI.D.)
Flow rate :1mL/min
Temp.
:40C
Detection :UV-VIS (210nm)

2
3
1

A
1

3
B

min

10

 Mobile Phase
Buffer Solution Notation and Preparation (1)
Preparation: 20mM Phosphoric acid buffer solution (pH2.5)

A : 20mM interpreted as phosphoric acid concentration

Mix 10mmol phosphoric acid and 10mmol sodium dihydrogen
phosphate, dissolve in 1L water

B : 20mM interpreted as sodium concentration

Dissolve 20mmol sodium dihydrogen phosphate in 1L water,
add phosphoric acid to adjust pH to 2.5

C : 20mM interpreted as phosphoric acid and sodium

concentrations, adjust pH with perchloric acid
Dissolve 20mmol sodium dihydrogen phosphate in 1L water,
add perchloric acid to adjust pH to 2.5

 Mobile Phase
Buffer Solution Notation and Preparation (2)
Mobile phase A: phosphoric acid interpreted as 20mM
Mobile phase B: sodium interpreted as 20mM
Mobile phase C: phosphoric acid, sodium both
interpreted as 20mM,
pH adjusted with
perchloric
acid
Column :Shim-Pack VP-ODS
1,

Peaks
1:
acetaminophen
2: dihydrocodeine
3: caffeine

(150mmL. x 4.6mmI.D.)
Flow rate :1mL/min
Temp.
:40C
Detection :UV-VIS (210nm)

3
A

If interpreted wrong
Dihyrocodeine elution position
is different!!

B
1

3
C

min

10

 Mobile Phase
pH Buffer Capacity and Peak Shape
Left figure: citric acid buffer solution (pH 4.5) used as mobile phase
Right figure: phosphoric acid buffer solution (pH 4.5) used as mobile phase
Column
Mobile
phase
Flow rate
Temp.
Detection

: STR ODS-II
(150mmL. x 4.6mm I.D.)
: 20mM buffer solution (pH 4.5)
/ acetonitrile (3/1, v/v)
: 1mL/min
: 40C
: UV-VIS (240nm)

If mobile phase buffer capacity is

weak
Peak shapes of acids and bases with pKa
near mobile phase pH deteriorate.

 Mobile Phase
Differences between Acetonitrile and Methanol
Absorbance HPLC grade acetonitrile has low absorbance at short UV wave
lengths

HPLC-grade acetonitrile good for hi-sens analysis at short UV wavelengths!

Column pressure For Acetonitrile, lower pressure is sufficient.

At same flow rate, extra pressure need not be applied to column.

Elution strength Acetonitrile generally stronger.

Some compounds strongly eluted with methanol. (carotene, cholesterol)

Selectivity Both show differences.

Peak shape Both show differences.
With polymer-type columns, acetonitrile is better.

Mobile phase degassing efficiency Caution when mixing with water

Methanol Exothermic degassing
Acetonitrile

Endothermic gas dissolution

 Mobile Phase
Effect of Flow Rate Change (1)
Retention time change
Peak area change Quantitation Error!!
Detector that responds absolute quantity of compound
Peak area is unaffected by flow rate.
Detector that responds to concentration of compound
Peak area is affected by flow rate.
*Almost all LC detectors are concentration-response type

 Mobile Phase
Effect of Flow Rate Change (2)
Peak Area

Area Ratio

A (1.00mL/min)

B (0.99mL/min)

B/A

methylparaben

3901107

3938213

1.0095

ethylparaben

2744605

2771087

1.0096

n-propylparaben

2626362

2652087

1.0098

n-butylparaben

2886547

2914591

1.0097

Column
: Shim-Pack VP-ODS (150mm L. x 4.6mm I.D.)
Mobile phase : methanol / water = 6/4 (v/v)
Flow rate
: A ; 1.00mL/min
B ; 0.99mL/min
Temperature : 40C
Detection
: UV-VIS (260nm)

 Mobile Phase

Flow Rate Change Influence in High Pressure Gradient Instrument

(1)

When mixing is performed with 2 pumps

Retention time is different from when mobile phase is pre-mixed in a bottle.

Peaks
1: methylparaben
2: ethylparaben
3: propylparaben

1
2

3
A : Pre-mixed solutions (2) delivered using one pump
B : Each solvent delivered with a separate pump and mixed

A
B

 Mobile Phase

Flow Rate Change Influence in High Pressure Gradient Instrument (1)

A : Pre-mixed solutions (2) delivered using one pump
B : Each solvent delivered with a separate pump and mixed
Peak Area

Area Ratio

B/A

methylparaben

4100127

4151876

1.013

ethylparaben

2885602

2922556

1.013

n-propylparaben

2761054

2797180

1.013

n-butylparaben

3035538

3075259

1.013

Column
Mobile phase
Flow rate
Temperature
Detection

: Shim-Pack VP-ODS (150mmL. x 4.6mm I.D.)

: methanol / water (6/4, vol/vol)
: 1.00mL/min
: 40C
: UV-VIS (260nm)

 Mobile Phase
Troubles caused by Bubbles Formed in Mobile Phase
Causes of bubble occurrence
Occurs when dissolved air exceeds air dissolution saturation point.
Causes: Solution warming, decreasing pressure, solvent mixing, etc.

Possible Troubles caused by bubbles

Mobile phase, pump
Retention time fluctuation, unstable solvent delivery

Column
Peak deformation, column degradation

Detector
Noise generation

Countermeasure: Offline and online degassing

However, the problems caused by air does not just come from
bubbles, but from dissolved air as well.

 Mobile Phase
Influence of Dissolved Air in Mobile Phase on Detection
Fluorescence detection
Quenching due to oxygen
Changes in peak area

Absorption detection
Changes in background absorbance
Baseline fluctuation (especially with gradient)
Appearance of peaks originating from oxygen

Refractive index detection

Baseline fluctuation

 Mobile Phase
Degassing Influence in Fluorescence Detection (1)

Mobile phase degassing may affect fluorescence intensity.

400

Degassed

mAU

* Analysis of bisphenol A
by fluorescence detection

BPA

300

Not degassed

Degassing : Helium purging

200

100

0.0

0.5

1.0

1.5

2.0

2.5

min

3.0

3.5

4.0

4.5

5.0

 Mobile Phase
Degassing Influence in Fluorescence Detection (2)
Gas-liquid membrane separation degasser takes time
to get degassing result.
Peak Area Value (106)

Analyte : Pyrene
Detection : Fluorescence
(Ex 310nm, Em 390nm)
Degassing:Gas-liq. separation membrane

4
3
2

Degasser OFF

Column

: Shim-Pack VP-ODS
(150mm L x 4.6mm I.D.)
Mobile Phase: acetonitrile/H2O = 4/1 (v/v)
Flow rate
: 1mL/min
Temperature : 40C

Degasser ON

1
0

0 10 20 30 40
Time from start of degassing (min)

50

60

 Mobile Phase
Background Changes in UV Detection

If dissolved oxygen concentration becomes high, organic solvent

absorbance becomes high.
This effect is pronounced in short wavelength region.

Air saturation
Degassed

Methanol Absorption Spectrum

 Mobile Phase
Dissolved Air-derived Peaks in UV Detection
Peak appears with mobile phase solution injection!?
The difference in dissolved air quantities in mobile phase and
sample solvent solutions can be expressed as peaks.

Mobile phase
not degassed

Degassed
mobile phase
Oxygen bubbling
Oxygen bubbling

Air saturation

Air saturation

He bubbling

He bubbling

Detection: UV210nm

 Mobile Phase
Dissolved Air-derived Peaks with RID Detector
15

0.0

mRIU

10

mRIU

- 0.2

- 0.4

Magnified
0

10

15

20

25

30

min

The difference in dissolved air quantities in

mobile phase and sample solvent solutions
can be expressed as peaks.

10

15

20

25

30

min

Column
: SCR-101C
Mobile phase : Water
Flow rate
: 0.5mL/min
Temperature : 85C
Detection
: RID detector
Degasser
: Gas-liq. Membrane Separ. (DGU-14A)
Sample
: 0.5% mannitol

 Detection
Cell Temperature Influence in UV Detection (1)
In general, spectrum shape is affected by ambient temperature.
Spectrum at low
temperature

Absorbance

Spectrum at high
temperature
Absorbance difference due to
temperature change

Measurement wavelength

 Detection
Cell Temperature Influence in UV Detection (2)
Comparison of peak area values without temperature control (34C)
Peak Area Ratios Compared with Areas at 34C

40C

50C

Phenol

0.994

0.991

Benzoic acid

0.991

0.986

p-toluic acid

0.985

0.968

Column
: Shim-Pack VP-ODS (150mm L. x 4.6mm I.D.)
Mobile phase : 10mM acetic acid (sodium) buffer solution (pH 4.5) /
Methanol = 7/3, (v/v)
Flow rate
: 1mL/min
Temperature : 40C
Detection
: UV-VIS (260nm)

 Detection
Cell Temperature Influence in Fluorescence Detection (1)
Normally, the higher the temperature, the lower the fluorescence intensity.
Analysis result of acridine (Temp. effect on fluorescence intensity)

Areas 2025C
13.5% decrease

Without temperature control (RF-10AXL)

Areas at 2025C
2.2% decrease

With temperature control (RF-10AXLsup

 Detection
Cell Temperature Influence in Fluorescence Detection (2)
Peak Area

20C

40C

Area Ratio
A40/A20

ASP

1500427

1165757

0.78

THR

1478447

1143925

SER

1570743

GLU

Peak Area

20C

40C

Area Ratio
A40/A20

TYR

1156850

850976

0.74

0.77

PHE

1315479

995832

0.76

1204273

0.77

HIS

1819675

1431665

0.79

1315462

1017768

0.77

LYS

487433

336837

0.69

PRO

505373

436522

0.86

ARG

1105530

839183

0.76

GLY

2440772

1924900

0.79

ALA

1558560

1203529

0.77

CYS

177078

138426

0.78

VAL

1565440

1226042

0.78

MET

1026839

790013

0.77

ILE

1457340

1126683

0.77

LEU

1185514

911017

0.77

Instrument :Shimadzu Amino Acid Analysis System

(Post-column derivitization using
o-phthaladehyde)
Detection :RF-10AXL Super
(Ex 350nm, Em 450nm)
(Cell temperature ; 20C, 40C)

 Detection
Solvent Influence on Fluorescence Intensity
Fluorescence Spectrum of Bisphenol A in Each
Solvent
(Excitation wavelength 270nm, with background
correction)

Methanol

Acetonitrile
Wavelength(nm)

 Sample

Curbing Decomposition of Components by Cooling

Cooling
No cooling

Change in Ascorbic Acid Peak Area

Without cooling, ascorbic acid

gradually decomposes.

Column
Mobile
phase

:SCR-101N (250mmL. x 7.9mm I.D.)

:10mM oxalic acid (sodium) buffer
solution (pH 3.8) including
1mM EDTA2Na

Flow rate
Temp.
Detection

: 1mL/min
: 40C
: UV-VIS (245nm)

 Sample
Influence of Sample Solvent on Peak Shape (1)

Theoretical plate number changes with different sample solvent.

Aqueous solvent

Methanol solvent
Analyte
Liquid flow

Methanol
solvent
Theoretical
Plate
Number

Sample: 10ul caffeine

Column:
Shim-pack CLC-ODS
(150mmL. x 6mm I.D.)
Mobile phase:
Methanol / H2O =3/7
Flow rate: 1mL/min
Temp. : Room temp.
Detection: 270nm

Aqueous solvent
Analyte
Liquid flow

Methanol / H2O

*Reversed phase chromatography with water /

methanol mobile phase mixture
Injection volume

 Sample
Influence of Sample Solvent on Peak Shape (2)
If sample solvent elution is stronger than that of mobile phase, peak shape deteriorates.

Column

: STR ODS-II
(150mm L. x 4.6mm I.D.)
Mobile
: 20mM citric acid (sodium) buffer
Phase
: solution (pH 4.5)
/ acetonitile = 3/1 ( v/v)
Flow rate : 1mL/min
Temp.
: 40C
Detection : UV-VIS (240nm)

Left Fig. : Sample solvent - water / acetonitrile

= 3/1 (v/v)
Right Fig.: Sample solvent - acetonitrile

 Sample
Advantages of Automated Sample Pretreatment

Improves analytical accuracy

Difficult to perform offline processing operations
completely uniformly, possibly causing poor
accuracy

Improves ease of operation

Offline processing may require special skills

Improves processing efficiency

Automated operation can be performed during
night, greatly improving processing efficiency

 Sample
Column Switching HPLC System
for Direct Injection of Blood Plasma (Co-Sense for BA)
Analytical column

Detector

Mobile phase
Autosampler

Inner Surface Reversed Phase

Pretreatment Column
Shim-Pack MAYI-ODS

Pump

Dilution bypass

Polymer like protein

Drug
Coating film
Image of drug and inner surface

Pump

Mobile phase

 Sample
Automated Pretreatment Example using Co-Sense for BA
Analysis of Plasma Spiked with Isopropylantipyrine
protein

Isopropylantipyrine

8.5
Time (min)

Injection volume
Mobile phase (Smpl. Inj. side)
Detection

: 100L
: 0.1% phosporic acid / acetonitrile = 95/5 (v/v)
: Isopropylantipyrine 275nm
Plasma matrix 280nm

17

 Sample

Comparison of Recovery by Pretreatment Methods

Recovery rate comparison after each of 2mg/mL ketoprofen and naproxen are
added to plasma (50mLinj.)
Manual Pretreatment
(Acetonitrile Added)

Automatic Pretreatment
Using Co-Sense for BA

 Sample
Causes and Control of Carryover
Causes and Means of Control in Autosampler
Cause: Adsorption to metallic materials due to ionic
interaction or coordination isomerism interaction (ionic
compounds or basic compounds)
Remove adsorbed components by needle washing
Control adsorption by making needle inert

Cause: Adsorption by hydrophobic interaction with resinous

materials (fat soluble compounds)
Wash rotor seal groove with organic solvent
Minimize adsorption by changing rotor seal material

 Sample
Needle Wash Mechanism (Total Volume Injection)
Sample loop
Needle

Wash port
Pump

Column

Ready
Readystate
state

Needle

Washing
Washingstate
state
Washing this surface is important.
Needle seal

 Sample
Selection of Needle Wash Solution
To control adsorption of substances due to ionic
interaction or coordination isomerism (ionic compounds
or basic compounds)
Acidify.
Make ion pair with large radius counter ion.
(Example) 100mM perchloric acid aqueous solution /
methanol (or acetonitrile)

Adsorption of compound due to hydrophobic interaction

(resinous compounds)
Wash with organic solvent.
* (Example) 100% methanol, acetonitrile, THF, etc.

 Sample
Minimizing Contamination due to Basic Substances (1)

Analyte : Chlorohexidin (basic compound)

Inject 2L of standard solution (mobile phase with 1.2mg/ml dissolved
compound), continuously inject 2L of mobile phase, compare areas.
Relative Peak Area
Carryover [%]

Standard
Solution

Blank

W/ Wash

0.000730

0.0730

W/O Wash

0.000425

0.0425

Needle material : SUS

Wash solution : Mobile phase
Needle wash process :
1.Immerse in wash port 3 sec.
2.Draw sample solution.
3.Immerse in wash port 3 sec.
4.Inject.

Column
Shim-pack VP-ODS (150mmL.x 2.0mm I.D.)
Mobile phase 10mM phosphoric acid buffer solution (pH 2.6) containing
100mMNaClO4 / acetonitrile

= 55 / 45 (v/v)
Flow rate
0.2mL/min
Temperature 40C
Detection
UV (260nm using semi micro cell)

 Sample
Minimizing Contamination due to Basic Substances (2)

Comparison using variously processed SUS needles

to make them inert
Relative Peak Area
Carryover [%]

Standard
Solution

Blank

SUS

0.000425

0.0425

Teflon coating

0.000023

0.0023

PEEK coating

0.000021

0.0021

Special metallic coating

0.000009

0.0009

Immersed in wash solution 3 sec. Other conditions same as previous page.

 Sample
Minimizing Contamination Due to Basic Substances (3)

In analysis of chlorohexidin (basic compound), carryover decreases from 0.07%

0.001% using needle immersion washing and a special metallic coating on
the needle surface!!

(Varies with component type, concentration and injection volume)

Chromatogram with injection of 2L standard solution

Chromatogram with injection of 2L mobile phase

chlorohexidin

chlorohexidin

* Ordinate full scale differs in the chromatograms.

 Sample
Minimizing Contamination of Hydrophobic Substances (1)
Sample loop

Pump

Needle

Column

Rotor seal

Adsorption of hydrophobic
compounds is thought to occur
mainly in rotor seal groove
through which samples pass.
It is believed that because the
adsorptive substance is washed
with mobile phase, it elutes
gradually.

 Sample
Minimizing Contamination of Hydrophobic Substances (2)

Analyte : Vitamin A Acetate

Inject 10L of standard solution 10g/mL: methanol dissolution ,
then inject 10L of blank solvent (methanol), and compare peak areas.
Relative Peak Area
Standard

Blank

[%]

10 minutes aging

0.000051

0.0051

20 minutes aging

0.000136

0.0136

Column

: Shim-Pack FC-ODS (75mmL.x

4.6mmI.D.
Mobile phase : (A) ; Water , (B) ; Methanol
0-2min, B solution 75%, hold
2-5min, B solution 75-100%, linear gradient
5-10min, B solution 100%, hold
Flow rate
: 0.5mL/min
Temperature
: 50C
Detection
: UV (325nm)

 Sample
Minimizing Contamination of Hydrophobic Substances (3)

By changing to a rotor seal made of PEEK, even after 20 minutes of

aging, almost no carryover was observed.

Chromatogram with injection of 10L standard solution

1.5

1.0

9412178

0.5

0.0

10

12

14

min

* Ordinate full scale differs in the chromatograms

Chromatogram with injection of 10L of blank

 Sample
High-Throughput Autosampler SIL-HT

Reduced carryover

Optimized materials for needle, rotor seal, etc.

Equipped with rinse mode

Multi-sample processing

2 sample trays / 4 micro-titer plates

Maximum sample number:

350 samples with 1.0mL vials
1536 samples with four 384-well micro-titer plates

High-speed sample injection

15 seconds required for sample injection (with 10 L injection)!

Equipped with high-performance measurement device

Total volume injection mode

Ideal LC autosampler for MS front end