liquid chromatography system

1. Important Notices for Daily Use of HPLC
Analytical Applications Department
Shimadzu Corporation

2. 2
 Important notice for Mobile Phases
 Important notice for Sample Solvents
 Important notice for Column Temperature
 Important notice for Detection
Contents

3. 3
Important Notice for Mobile Phases

4. 4
 Water contains a lot of other things, including:
- Organic and inorganic substances
- Ionic and nonionic substances
- UV adsorbent and fluorescent substances
Selecting Water (1)
- Lab-made
… distilled water, ion-exchange water, ultrapure water, etc.
(Ultrapure water: treated by reverse osmosis, ion-exchange
membrane, membrane filter, UV irradiation, etc.)
- Commercial
… distilled water, ion-exchange water, purified water, pure
water, HPLC-grade distilled water, etc.

5. 5
 Example peaks in a blank gradient with
no sample injection
Selecting Water (2)
Peaks of impurities in water
Water impurities retained in the column are eluted by acetonitrile.
100% acetonitrile
100%
water

6. 6
 What sort of water should we select?
Distilled water, ion-exchange water, purified water,
pure water, ultrapure water …
Selecting Water (3)
HPLC-grade distilled water … reduces absorbance
in short UV wavelength region
Gradient elution method for reversed-phase HPLC
Less baseline drift and ghost peaks
Ultrapure water … reduces all impurities
General HPLC, ion chromatography
Safe to use for many purposes

7. 7
 Clean water is easier to be contaminated
Selecting Water (4)
Restrict the causes of contamination
Examples: work environment, equipment, handling
methods, etc.
Equipment producing ultrapure water
needs strict maintenance
Examples: reverse osmosis membrane,
ion-exchange cartridges, etc.
Take care with storage containers
Examples: leaching of UV-absorption agents from
polyethylene containers

8. 8
Selecting Organic Solvent (1)
 Acetonitrile and methanol …absorbance
HPLC-grade acetonitrile has low absorbance
Methanol
Acetonitrile
Low absorbance in short
wavelength region
HPLC-grade acetonitrile is good for analysis in the short (UV)
wavelength range.
Special grade
Special grade
HPLC grade
Wavelength (nm)
Wavelength (nm)
Absorbance
(AU)
Absorbance
(AU)

9. 9
Acetonitrile has
- Low viscosity
Lower column pressure load
at same flow rate
- Small viscosity change when
mixed with water
Small column pressure
changes during gradient analysis
It is soft to the column.
 Acetonitrile and methanol … viscosity
Selecting Organic Solvent (2)
Water/methanol
Water/methanol
Water/acetonitrile
Water/acetonitrile
Pressure
(MPa)
Water (100%) Organic solvent (100%)
Water/Organic solvent 1:1

10. 10
 Acetonitrile and methanol … elution
However, the elution power relationship may reverse with high proportions
of organic solvent. (right diagram)
Characteristics of a protic solvent (methanol) and an aprotic solvent (acetonitrile)
Generally, acetonitrile has higher elution power
Methanol25/water75
Acetonitrile10/water90
Methanol95/water 5
Acetonitrile95/water 5
試料:コレステロール
試料:カフェイン
Selecting Organic Solvent (3)
Sample: caffeine Sample: cholesterol

11. 11
Acetonitrile is safe to use for most analyses.
Use methanol for poor selectivity or peak
shape.
Try methanol for samples with poor solubility
in acetonitrile.
Selecting Organic Solvent (4)
 Acetonitrile and methanol … Which one to select?

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• Water/Methanol = 1/1 (v/v)
– Measure 500 mL water and 500 mL
methanol separately and mix them
together.
– Total does not make 1 L.
• 50% (v/v) Methanol water solution
– Put 500 mL methanol in a 1L volumetric flask
and add water to make 1 L.
– Higher proportion of water
1L
Water 500 mL Methanol 500 mL
Methanol 500 mL
Water
 Water/organic solvent mixing methods
and notation
Mobile Phase Preparation (1)

13. 13
 Different results from different mixing methods
1
2
3
3
1
A
B
2
Peaks
1: Acetaminophen
2: Dihydrocodeine
3: Caffeine
0 2 4 6 8 10
min
Mobile phase A:
20 mmol/L Sodium(phosphate) buffer
<pH 2.5>/Acetonitrile = 9:1 (v/v)
Mobile phase B:
20 mmol/L Sodium(phospate) buffer
<pH 2.5> containing 10% (v/v) acetonitrile
Mobile Phase Preparation (2)

14. 14
How do we make 20 mmol/L sodium phosphate buffer (pH2.5)?
A: If “20 mmol/L” is considered as the phosphate concentration.
Mix 10 mmol phosphoric acid and 10 mmol sodium dihydrogenphosphate and dissolve in 1
L of water.
B: If “20 mmol/L” is considered as the sodium concentration.
Dissolve 20 mmol sodium dihydrogenphosphate in 1 L of water and then drop phosphoric
acid to adjust pH to 2.5.
C: If “20 mmol/L” is considered as the phosphate and sodium concentration pH is adjusted
with perchloric acid.
Dissolve 20 mmol sodium dihydrogenphosphate in 1 L of water and then drop perchloric
acid to adjust pH to 2.5.
 Preparation of buffer solution and notation
Mobile Phase Preparation (3)

15. 15
 Different results from different buffer preparation methods
Mobile phase A: considered as
20 mmol/L phosphoric acid
Mobile phase B: considered as
20 mmol/L sodium
Mobile phase C: considered as
20 mmol/L phosphoric acid and
20 mmol/L sodium
min
1 2 3
3
1
A
B
Peaks
1: Acetaminophen
2: Dihydrocodeine
3: Caffeine
2 4 6 8 10
C
1+2
3
2
0
Mobile Phase Preparation (4)

16. 16
1)100 mmol/L (sodium) phosphate buffer (pH2.1)
- Sodium dihydrogenphosphate dihydrate (MW=156.01) 50 mmol (7.8 g)
- Phosphoric acid (85%, 14.7 mol/L) 50 mmol (3.4mL)
- Dissolve in water to make 1L.
2)10 mmol/L (sodium) phosphate buffer (pH2.6)
- Sodium dihydrogenphosphate dihydrate (MW=156.01) 5 mmol (0.78 g)
- Phosphoric acid (85%, 14.7 mol/L) 5 mmol (0.34 mL)
- Dissolve in water to make 1L.
- Alternatively, dilute 1) 10 times.
 Preparation of phosphoric acid buffer without using a pH meter
Mobile Phase Preparation (5)

17. 17
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. × 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 phase
Right figure: phosphoric acid buffer solution (pH 4.5) used as mobile phase
pH Buffer Capacity and Peak Shape

18. 18
Changes in absorbance dependent on the solvent
 Changes in absorbance due to dissolved air
Effects of Dissolved Air (1)
Methanol
THF
Hexane
Acetonitrile
Water
Spectra of Increased Absorbance Due to Air Saturation

19. 19
Column: ODS
Mobile phase: Methanol/water (85/15, v/v)
Caused by differences in dissolved air volume in
the mobile phase and sample solution due to
degassing the mobile phase.
 Appearance of peaks due to dissolved air
With mobile
phase
degassing
Without mobile
phase
degassing
Flowrate: 1.0 mL/min
Detection: UV 210 nm
Effects of Dissolved Air (2)
O2 bubbling
Air saturation
He bubbling
O2 bubbling
Air saturation
He bubbling

20. 20
 Confirming peaks due to dissolved air
Effects of Dissolved Air (3)
Mobile phase
Pump
Injector Column oven
Column
Detector
Degasser
Air mixed in Degassed
Sample solution
Air saturation in mobile phase and sample solution
Check by injecting degassed effluent
Dissolved air is injected

21. 21
 Measures to deal with peaks due to dissolved air
Effects of Dissolved Air (4)
Change the type of organic solvent
If methanol is used, change to acetonitrile that has
little change in absorbance due to dissolved air.
Change the mobile phase composition
Lower the organic solvent ratio (particularly with
methanol).
Change the column
Use a column with a smaller retention capacity.

22. 22
min
0.0 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
400
BPA
<< With degassing
<< Without degassing
Analysis of Bisphenol A by Fluorescence Detection
 Quenching phenomenon in fluorescence detection
Degassing results in increased
fluorescence intensity
Effects of Dissolved Air (5)
Quenching occurs due to dissolved oxygen

23. 23
Important Notice for Sample Solvents

24. 24
Peak shape changes due to solvent composition
Peak shape deteriorates as injection volume increases
Appropriate choice of sample solvent?
 Sample solvent composition and injection volume
Problems due to Sample Solvents

25. 25
 Change in peak shape due to sample solvent
Left: Sample solvent = water/acetonitrile (3/1, v/v)
Right: Sample solvent = acetonitrile
Column : STR ODS-II (150mm L. x 4.6mm i.d.)
Mobile phase: 20 mmol/L citric acid-sodium buffer (pH
4.5)/Acetonitrile (3/1, v/v)
Flowrate : 1.0 mL/min
Temperature : 40ºC
Detection : UV240 nm
Peaks spread!
Sample Solvents and Peak Shape

26. 26
Reversed phase with water/methanol mixture as mobile phase
Mobile phase flow
Methanol solvent
Sample components
Mobile phase flow
Water solvent
Sample components
 Behavior of sample solvent and solute in the column
Analyte band spreads out in methanol, which has
large elution power
Sample Solvents and Analyte Band

27. 27
 Difference in elution power affects the theoretical plate number
High injection volumes
possible with low-elution-
power sample solvent
Take care if the sample
solvent has higher elution
power than the mobile phase.
Sample Solvents and Injection Volume (1)
Sample solvent: water
Sample solvent: methanol /water = 3/7
(same as mobile phase)
Sample solvent:
methanol
HPLC conditions
Sample: caffeine 10 ug
Column: Shim-pack CLC-ODS
(150 mmL x 6 mm ID)
Mobile phase: methanol/water=3/7
Flow rate: 1 mL/min
Temp: room temp.
Detection: 270 nm
Theoretical
plate
number
Injection volume uL

28. 28
 Peaks deform due to injection volume
Peak shape can be improved by reducing
the injection volume
Sample Solvents and Injection Volume (2)
Elution starts from around 3 min.
when 100 uL is injected. HPLC conditions
Same as previous slide
Analyte caffeine
Sample solvent: methanol

29. 29
 Adsorption of basic substances onto glass surface
Beware of losses due to adsorption onto vial
Sample solvent
Aqueous solution of perchloric acid Water
No. of analy sis caffeine thiamine caffeine thiamine
1st 50248 52974 50327 48228
2nd 50120 52691 50372 48293
3rd 50268 53031 50479 48434
4th 50320 52801 50259 48060
5th 50443 52722 50224 47951
ave 50280 52844 50332 48193
RSD(%) 0.23 0.29 0.20 0.40
Sample Solvents and Adsorption
Sample solvents : a) aqueous solution of perchloric acid, b) water
Sample concentration : caffeine, thiamine both 10 mol/L
Injection volume : 10 L

30. 30
Important Notice for Column Temperature

31. 31
Analysis at constant temperature
Enhanced accuracy (repeatability, reproducibility)
Effects of Column Temperature Control
Enhanced column efficiency
Decreased column pressure
Enhanced sample solubility, etc.
Analysis at higher temperature
Analysis at lower temperature
Enhanced selectivity (in particular case)
Enhanced sample stability, etc.

32. 32
Different behaviors of components
Column Temperature and Selectivity
Peaks
1. Benzoic Acid
2. Sorbic Acid
3. Methylparaben
30C
25C
20C
Sometimes separation can be
controlled without changing the
mobile phase composition.

33. 33
Anomer separation in the column
Separation of sugar anomers by the ligand-exchange method
Column Temperature and Peak Shape (1)
Peaks
1. Sucrose
2. Glucose
3. Sorbitol
Column : SCR-101C (Ca type)
Mobile phase : water
Flowrate : 0.8 mL/min
Detection : differential refractive index
Increasing the column temperature and
increasing the anomer equilibration
speed forms a single peak.
Room temp. (25C)

34. 34
Temperature gradient in the mobile phase in the column
Heating creates a broad peak.
Effect increases at larger column I.D. and mobile phase
flow rate
<Countermeasure> Connect a preheat coil before the column inlet or auto-sampler.
Analyte band spreads out
Temperature gradient in the column
Column Temperature and Peak Shape (2)

35. 35
0 0.5 1 1.5 2 2.5 min
0 0.5 1 1.5 2 2.5 min
Without pre-heat coil
Column : ODS (50mmL. x 3 mm ID)
Mobile phase : water/acetonitrile (3/2, v/v)
Flowrate : 1.0 mL/min
Effects of the pre-heat coil
With pre-heat coil
Temperature : 50C
Detection : UV 260 nm
Sample : parabens
Column Temperature and Peak Shape (3)

36. 36
Important Notice for Detection

37. 37
Effects of instrument environment
Detection and Temperature
In a room with high temperature fluctuations or near
an airconditioner outlet
Generally, refractive index detectors, conductivity detectors, and
electrochemical detectors are sensitive to temperature effects,
causing baseline drift.
But UV detectors and fluorescence detectors
are also affected by temperature fluctuations.

38. 38
- Fluorescence intensity decreases as the temperature rises.
- Fluorescence intensity fluctuates due to temperature fluctuations.
As the temperature increases, intermolecular collisions
increase, resulting in losses in potential energy
Fluorescence Intensity and Detection Temperature
Maintain the room temperature as constant as possible.
>> Enhanced repeatability
Set the detection temperature as low as possible.
>> Enhanced sensitivity
Temperature in Fluorescence Detection (1)

39. 39
Room-temperature fluctuations and detector cell
inlet temperature
Temperature in Fluorescence Detection (2)
Changes in cell inlet temperature due to room temperature fluctuations (at 1 mL/min. flow rate)
Time (minutes)
Temperature
(C)
Room temperature
Liquid temperature
(no temp. control)
Liquid temperature
(with temp. control)

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Effects of room-temperature fluctuations and
cell temperature control
Area value decreases
13.5％
from 20°C to 25°C
Without cell temp. control With cell temp. control
Component: acridine
At 20
At
25°C
At 20°C, 25°C
Time (minutes)
Area value decreases
2.2％
from 20°C to 25°C
Time (minutes)

169. 169
Effects of room temperature fluctuations
on mobile phase absorbance
Baseline stabilization due to cell temperature
control of UV detector
Temperature for UV Detection (1)
Mobile phase: phosphoric acid–sodium buffer solution (pH 7), detection :UV 210 nm
Without cell temperature control
Cell temp. Cell temp.
Room temp.
Baseline
Room temp.
Baseline
With cell temperature control

170. 170
Enhanced precision due to cell temperature control
0 5 10 min
0 5 10 min
Without cell
temp. control
With cell temp.
control
Column : ODS (150 mm x 4.6 mm ID)
Mobile phase : phosphoric acid buffer (pH
7)/Methanol (3/1, v/v)
Flow rate : 1.0 mL/min
Temperature : 40C
Detection : UV-VIS detector with
temperature control (210 nm)
Sample : caffeine (1 ng)
Without cell
temp. control
With cell temp.
control
1st 1.000 1.000
2nd 0.987 0.999
3rd 0.963 1.008
4th 0.992 0.994
5th 1.031 0.995
6th 1.026 1.008
RSD (%) 2.55 0.61
Temperature for UV Detection (2)

171. 171
Detector Response (1)
0 0.5 1 1.5 min 0 0.5 1 1.5 min 0 0.5 1 1.5 min
Response: 50 ms
Theoretical plate
number: 2642 (Peak 4)
Noise: 21.1 AU
Response: 500 ms
Theoretical plate number:
2426 (Peak 4)
Noise: 5.2 AU
Response: 1500 ms
Theoretical plate number:
1400 (Peak 4)
Noise: 0.5 AU
Column : ODS (50 mmL. x 3 mm ID)
Mobile phase : Water/acetonitrile (1/1, v/v)
Flow rate : 1.0 mL/min
Peak shape changes due to response
Temperature : 25°C
Detection : UV260 nm
Sample : parabens

172. 172
Response setting according to peak width
Detector Response (2)
Response Suitable peak width
50 ms Unlimited
100 ms 1s or more
500 ms 4s or more
1000 ms 7s or more
1500 ms 10s or more
0.8
1
1.2
1.4
1.6
1.8
2
2.2
3 4 5 6 7 8
Peak Width /s
Observed
Peak
Width
(normalized)
50 ms
100 ms
500 ms
1000 ms
1500 ms
“1.1” line with peak
width at 50 ms set to “1”

173. 173
Conclusions
Important Notice for Mobile Phases
Selecting Water / Organic Solvent; Mobile Phase Preparation; Effects of
Dissolved Air
Important Notice for Sample Solvents
Sample Solvents and Peak Shape; Theoretical Plate Number
Important Notice for Column Temperature
Effects of Column Temperature Control; Column Temperature and
Selectivity / Peak Shape
 Important Notice for Detection
Detection and Temperature; Detector Response