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 ）

• 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

For better analysis in HPLC…

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 25ºC.

• water/ethanol=1/1 (v/v)

– Measure 500mL each of water and ethanol, and then mix

– The total is not 1L.

• 50%(v/v) ethanol aqueous sol.– Transfer 500mL ethanol to 1L

volumetric flask, bring to volume with water.

– The water percentage is high.

1LWater Ethanol

EthanolWater

Mobile Phase Solvent Mixing Ratio Notation and Preparation (2)

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>

1

2

3

31

A

B

2Peaks 1: acetaminophen 2: dihydrocodeine 3: caffeine

0 2 4 6 8 10min

Column :Shim-Pack VP-ODS (150mmL. x 4.6mmI.D.)

Flow rate :1mL/min

Temp. :40ºC

Detection :UV-VIS (210nm)

Mobile Phase Solvent Mixing Ratio Notation and Preparation (3)

• Preparation: 20mM Phosphoric acid buffer solution (pH2.5)

Mobile Phase Buffer Solution Notation and Preparation (1)

• 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

Column :Shim-Pack VP-ODS (150mmL. x 4.6mmI.D.)

Flow rate :1mL/min

Temp. :40ºC

Detection :UV-VIS (210nm)

Mobile phase A: phosphoric acid interpreted as 20mMMobile phase B: sodium interpreted as 20mMMobile phase C: phosphoric acid, sodium both

interpreted as 20mM, pH adjusted with perchloric acid

Mobile Phase Buffer Solution Notation and Preparation (2)

min

1

2

3

31

A

B

2

Peaks 1: acetaminophen 2: dihydrocodeine 3: caffeine

0 2 4 6 8 10

C

3

1,2

If interpreted wrong…Dihyrocodeine elution position is different!!

If mobile phase buffer capacity is weak…Peak shapes of acids and bases with pKa near mobile phase pH deteriorate.

Column : STR ODS-II (150mmL. x 4.6mm I.D.)

Mobile : 20mM buffer solution (pH 4.5)phase / acetonitrile (3/1, v/v)

Flow rate : 1mL/min

Temp. : 40ºC

Detection : UV-VIS (240nm)

Left figure: citric acid buffer solution (pH 4.5) used as mobile phaseRight figure: phosphoric acid buffer solution (pH 4.5) used as mobile phase

Mobile Phase pH Buffer Capacity and Peak Shape

• 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 Differences between Acetonitrile and Methanol

• 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 (1)

1.009729145912886547n-butylparaben

1.009826520872626362n-propylparaben

1.009627710872744605ethylparaben

1.009539382133901107methylparaben

B/AB (0.99mL/min)A (1.00mL/min)

Area RatioPeak Area

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 : 40ºC

Detection : UV-VIS (260nm)

Mobile Phase Effect of Flow Rate Change (2)

When mixing is performed with 2 pumps…

A

B

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

Peaks 1: methylparaben 2: ethylparaben 3: propylparaben1

2

3

Mobile Phase Flow Rate Change Influence in High Pressure Gradient Instrument

(1)

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

1.01330752593035538n-butylparaben

1.01327971802761054n-propylparaben

1.01329225562885602ethylparaben

1.01341518764100127methylparaben

B/ABA

Area RatioPeak Area

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

Mobile phase : methanol / water (6/4, vol/vol)

Flow rate : 1.00mL/min

Temperature : 40ºC

Detection : UV-VIS (260nm)

•A : Pre-mixed solutions (2) delivered using one pump

•B : Each solvent delivered with a separate pump and mixed

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

• 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 Troubles caused by Bubbles Formed in Mobile Phase

• 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 Influence of Dissolved Air in Mobile Phase on Detection

min0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

mAU

0

100

200

300

400BP

A

Degassed

Not degassed

* Analysis of bisphenol A by fluorescence detection

Degassing : Helium purging

Mobile Phase Degassing Influence in Fluorescence Detection (1)

• Mobile phase degassing may affect fluorescence intensity.

• Gas-liquid membrane separation degasser takes time to get degassing result.

0

1

2

3

4

5

0 10 20 30 40 50 60Time from start of degassing (min)

Pea

k A

rea

Val

ue

(×

106 )

Degasser OFF

Degasser ONColumn : Shim-Pack VP-ODS

(150mm L x 4.6mm I.D.)Mobile Phase: acetonitrile/H2O = 4/1 (v/v)Flow rate : 1mL/minTemperature : 40ºC

Analyte : Pyrene

Detection : Fluorescence (Ex 310nm, Em 390nm)

Degassing:Gas-liq. separation membrane

Mobile Phase Degassing Influence in Fluorescence Detection (2)

Methanol Absorption Spectrum

• If dissolved oxygen concentration becomes high, organic solvent absorbance becomes high.

• This effect is pronounced in short wavelength region.

Mobile Phase Background Changes in UV Detection

Air saturation

Degassed

• Peak appears with mobile phase solution injection!?

Mobile Phase Dissolved Air-derived Peaks in UV Detection

Detection: UV- 210nm

The difference in dissolved air quantities in mobile phase and sample solvent solutions can be expressed as peaks.

Degassed mobile phase

Mobile phase not degassed

Oxygen bubbling

Air saturation

He bubbling

Oxygen bubbling

Air saturation

He bubbling

min5 10 15 20 25 30

mRIU

0

5

10

15

min5 10 15 20 25 30

mRIU

- 0.4

- 0.2

0.0

Magnified

Mobile Phase Dissolved Air-derived Peaks with RID Detector

Column : SCR-101C

Mobile phase : Water

Flow rate : 0.5mL/min

Temperature : 85ºC

Detection : RID detector

Degasser : Gas-liq. Membrane Separ. (DGU-14A)

Sample : 0.5% mannitol• The difference in dissolved air quantities in

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

• In general, spectrum shape is affected by ambient temperature.

Measurement wavelength

Spectrum at high temperature

Absorbance difference due to temperature change

Spectrum at low temperature

Abs

orba

nce

Detection Cell Temperature Influence in UV Detection (1)

• Comparison of peak area values without temperature control (34C)

0.9680.985p-toluic acid

0.9860.991Benzoic acid

0.9910.994Phenol

50ºC40ºC

Peak Area Ratios Compared with Areas at 34ºC

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 : 40ºC

Detection : UV-VIS (260nm)

Detection Cell Temperature Influence in UV Detection (2)

With temperature control (RF-10AXLsuper)

Areas 20→25C13.5% decrease

Areas at 20→25C2.2% decrease

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)

Without temperature control (RF-10AXL)

0.779110171185514LEU

0.7711266831457340ILE

0.777900131026839MET

0.7812260421565440VAL

0.78138426177078CYS

0.7712035291558560ALA

0.7919249002440772GLY

0.86436522505373PRO

0.7710177681315462GLU

0.7712042731570743SER

0.7711439251478447THR

0.7811657571500427ASP

40ºC20ºC

Area RatioA40/A20

Peak Area Area RatioA40/A20

Peak Area

40ºC20ºC

0.768391831105530ARG

0.69336837487433LYS

0.7914316651819675HIS

0.769958321315479PHE

0.748509761156850TYR

Instrument : Shimadzu Amino Acid Analysis System (Post-column derivitization using o-phthaladehyde)

Detection : RF-10AXL Super (Ex 350nm, Em 450nm) (Cell temperature ; 20ºC, 40ºC)

Detection Cell Temperature Influence in Fluorescence Detection (2)

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

Detection Solvent Influence on Fluorescence Intensity

Methanol

Acetonitrile

Wavelength(nm)

750000

800000

850000

900000

1 2 3 4 5 6 7 8

No. of Analyses

Pea

k Are

a

冷却あり冷却なし

• Without cooling, ascorbic acid gradually decomposes.

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

Mobile :10mM oxalic acid (sodium) buffer

phase solution (pH 3.8) including 1mM EDTA•2Na

Flow rate : 1mL/min

Temp. : 40ºC

Detection : UV-VIS (245nm)

Change in Ascorbic Acid Peak Area

Sample Curbing Decomposition of Components by Cooling

CoolingNo cooling

Sample Influence of Sample Solvent on Peak Shape (1)

Liquid flow

Methanol solvent

Analyte

Liquid flow

Aqueous solvent

Analyte

*Reversed phase chromatography with water / methanol mobile phase mixture

• Theoretical plate number changes with different sample solvent.

Aqueous solvent

Methanol / H2O

Methanol solvent

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

Theoretical

Plate

Number

Injection volume

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

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. : 40ºC

Detection : UV-VIS (240nm)

Sample Influence of Sample Solvent on Peak Shape (2)

If sample solvent elution is stronger than that of mobile phase, peak shape deteriorates.

• 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 Advantages of Automated Sample Pretreatment

Sample Column Switching HPLC System

for Direct Injection of Blood Plasma (Co-Sense for BA)

Pump

Analytical column

Autosampler

Inner Surface Reversed Phase Pretreatment Column

Shim-Pack MAYI-ODS

Pump

Detector

Mobile phase

Mobile phase

● ●

Dilution bypass

Polymer like protein

Drug

Coating film

Image of drug and inner surface

0 8.5 17Time (min)

proteinprotein IsopropylantipyrineIsopropylantipyrine

Injection volume : 100μLMobile phase (Smpl. Inj. side) : 0.1% phosporic acid / acetonitrile = 95/5 (v/v)Detection : Isopropylantipyrine ； 275nm

Plasma matrix ； 280nm

Sample Automated Pretreatment Example using Co-Sense for BA

•Analysis of Plasma Spiked with Isopropylantipyrine

• 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

Ketoprofen Naproxen Ketoprofen Naproxenn=1 91.1 92.0 96.0 96.7n=2 96.6 96.3 95.7 97.0n=3 93.9 97.0 97.2 96.9n=4 92.4 94.1 97.3 97.0n=5 90.2 91.7 95.6 98.2

Average 92.8 94.2 96.4 97.2S.D. 2.5 2.4 0.8 0.6

C.V.(%) 2.7 2.6 0.9 0.6

Sample Comparison of Recovery by Pretreatment Methods

• 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

SampleCauses and Control of Carryover

Causes and Means of Control in Autosampler

Washing this surface is important.

NeedleWashing stateWashing stateReady stateReady state

Sample loop

Pump

Column

Wash portNeedle

Needle seal

Sample Needle Wash Mechanism (Total Volume Injection)

• 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 Selection of Needle Wash Solution

• 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.

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/minTemperature 　： 40ºCDetection 　　 ： UV (260nm ， using semi micro cell)

Sample Minimizing Contamination due to Basic Substances (1)

0.04250.0004251W/O Wash

0.07300.0007301W/ Wash

BlankStandard Solution

Carryover [%]

Relative Peak Area Needle material : SUSWash solution : Mobile phaseNeedle wash process :

1.Immerse in wash port 3 sec.2.Draw sample solution.3.Immerse in wash port 3 sec.4.Inject.

• Comparison using variously processed SUS needles to make them inert

0.00230.0000231Teflon coating

0.04250.0004251SUS

0.00090.0000091Special metallic coating

0.00210.0000211PEEK coating

BlankStandard Solution

Carryover [%]

Relative Peak Area

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

Sample Minimizing Contamination due to Basic Substances (2)

Chromatogram with injection of 2µL mobile phase

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 2µL standard solution

* Ordinate full scale differs in the chromatograms.

chlorohexidin

chlorohexidin

• 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 loop

Pump

Column

Needle

Rotor seal

Sample Minimizing Contamination of Hydrophobic Substances (1)

• Analyte : Vitamin A Acetate • Inject 10µL of standard solution （ 10μg/mL: methanol dissolution ） ,

then inject 10µL of blank solvent (methanol), and compare peak areas.

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/minTemperature : 50ºCDetection : UV (325nm)

Sample Minimizing Contamination of Hydrophobic Substances (2)

Relative Peak Area

Standard Blank

10 minutes aging 1 0.000051 0.0051

20 minutes aging 1 0.000136 0.0136

Ｃａｒｒｙｏｖｅｒ[%]

• By changing to a rotor seal made of PEEK, even after 20 minutes of aging, almost no carryover was observed.

Sample Minimizing Contamination of Hydrophobic Substances (3)

min0 2 4 6 8 10 12 14

V

0.0

0.5

1.0

1.594

1217

8

Chromatogram with injection of 10µL of blankChromatogram with injection of 10µL standard solution

* Ordinate full scale differs in the chromatograms

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