Practice and Ramifications of Ultrafast

Chiral &Achiral LC and SFC

Daniel W. Armstrong

Robert A. Welch Professor

University of Texas at Arlington

Department of Chemistry and Biochemistry

Arlington, TX 76019

This is accomplished in chiral and achiral LC separations in the same way:

a) By going to smaller more efficient supports

b) Using bonding chemistries that provide more efficient separations

c) Effective packing of small particles

? Ultrafast, High Efficiency LC Separations:Practice & Ramifications

What to choose, SPPs or sub 2 micron particles?

For SFC, pressure effects also must be considered!

One of the caveats of high efficiency and/or ultrafast separations is that you can exceed the

capabilities of current instrumentation.

1. We can now complete separations faster than conventional devices can inject samples (60-30 s).

2. Some detectors & settings can be inadequate.

3. Extra-column effects become critical.

4. Thermal effects become significant.

What Does Superficially Porous Mean?

• Improved mass transfer kinetics

• Low diffusion times

• Improved eddy dispersion

• Allows for high linear flow rates

Non-C18 columns that are either essential or superior for doing: HILIC, chiral, isomeric, isotope, peptide, etc. fast, high efficiency separations.

Some SPP Stationary Stationary Phases

Separation of fipronil enantiomers using an isopropylated cyclofructan 6 chiral selector bound to FPPs and SPPs. The mobile phase was heptane/ethanol (95/5). Note the increased resolution and efficiency when using the SPP-CSP. Also, the analysis time was considerably shorter for the separation performed using the SPP-CSP. The advantages are a result of the increased efficiency afforded by the SPPs, without a concomitant loss of selectivity.

FPP

SPP

Rs = 1.6

Rs = 2.1

We expected higher efficiencies and lower retention, but not necessarilyhigher resolution (less surface area = less chiral selector = lower selectivity).

Constant retention comparison of the enantiomeric separation of fipronil

using an isopropylated chiral selector bound to FPPs and SPPs. The mobile phase composition was changed to allow all compounds to have similar retention. For the FPP chromatogram the mobile phase was heptane/ethanol (92/8). For the SPP chromatogram the mobile phase composition was heptane/ethanol (95/5). The flow rate in both cases was 1.0 mL/min. This example clearly demonstrates the overall gains in separation performance when using the SPP-CSPs. Note the large increase in resolution for the SPP-CSP.

Fipronil

FPP

SPP

Even higher resolution!

Type

Particle

Size

(µm)

Pore

size

(Å)

Surface

Area

(m2/g)

C

(%)

N

(%)

µmol/m2 a

Selector

loadinga

(% )

FPP 5 115 465 14.1 1.1 0.77 32.2

SPP 2.7 120 120 6.2 0.88 0.88 13.1

Example of particle properties and elemental analysis for

CSPs produced on FPPs and SPPs. The chemistries are

identical. Even higher alphas observed. This is what is

necessary for the achievement of faster and higher Rs

separations!

Note, the much lower surface area for the SPP compared to the FPP. Yet, a

higher relative coverage (i.e. µmol/m2) of the “propyl-CF6” chiral selector is

actually obtained on the SPPs. a Values calculated starting with the % C

measured by elemental analysis.

Using 5-10 cm chiral columns nearly 100ultra-fast enantiomeric separations

were reported

Analytical Chemistry 87 (2015) 9137-9148.

What about difficultnon-chiral separations?

-1

19

39

59

79

99

0,0 5,0 10,0 15,0 20,0 25,0 30,0 35,0 40,0 45,0 50,0

Ab

s (A

MU

)

Time (min)

70/30 ACN/NH4HCO2 2.5 mM pH 3.7, 0.8 mL min-1 , 220 nm UV DAD detector, 100 x 4.6 mm i.d. , 2.7 µm SPP TeicoShell

75/25 ACN/water,1.0 mL min-1,

Commercial 5 µm FPP, 250 x 4.6 mm i.d.Chirobiotic T

B.Zhang et. al./ J Chromatogr A 1053 (2004) 89-99

0

1

2

3

4

0 5 10 15 20 25 30

Ab

s (A

MU

)

Time (min)

40/60 ACN/0.1% HCOOH,0.5 mL min-1,

Commercial 5 µm FPP, 250 x 4.6 mm i.d.Chirobiotic TAG

B.Zhang et. al./ J Chromatogr A 1053 (2004) 89-99

35/65 ACN/, NH4HCO2

50 mM, pH 3.33.5 mL min-1, 30 0C 280 nm2.7 µm SPP, 50 x 4.6 mm i.d. TeicoShell

R = Arg, K = Lys

Closely related fluoro and desfluoro compounds (Welch & co-workers at Merck indicated that some of these separations could be

more difficult than chiral)

Ultrafast Separation of ezetimibe, ciprofloxacin, ofloxacin and their desfluro analogues

hydroxylpropyl-β-cyclodextrin SPP (5

cm x 0.46 cm) column, MP: 50:50 5 mM

NH4OAc pH 4.0: MeOH

flow rate 2.0 mL/min

CF6 SPP (15 cm x 0.46 cm) column,

MP: 90:10:0.3:0.2 ACN/MeOH/TFA/TEA,

flow rate 4.5 mL/min

CF6 SPP (15 cm x 0.46 cm) column, MP: 90:10:0.3:0.2

ACN/MeOH/TFA/TEA, flow rate 4.5 mL/min

Optimized separation factors of fluoro and desfluoro compounds

Mixtures Columna Mobile phaseFlow rate

(mL/min)

t1

(min)

t2

(mi

n)

Selectivity

(α)

Voriconazole and

Desfluoro voriconazoleA

99:1:0.3:02

ACN/IPA/TFA/TEA2.00 0.71 1.04 2.73

Ciprofloxacin and

Desfluoro ciprofloxacinA

99:1:0.3:02

ACN/IPA/TFA/TEA2.00 2.91 4.29 1.58

Ofloxacin and Desfluoro

ofloxacinA

99:1:0.3:02

ACN/IPA/TFA/TEA2.00 1.44 1.83 1.42

a (A)10 cm × 0.46 cm column packed with CF6-P SPP

J. Chromatogr. A, 1426 (2015) 241-247.

On Selector-Based Bonded Brush-Type Phases:

•At the same mobile phase composition, SPP-CSPs have 20-40% higher resolution.

•At the same mobile phase composition, analytes are eluted 40-70% faster.

•At the same mobile phase composition, SPP-CSPs have approximately 2-5x the plate count.

•At constant retention, SPP-CSPs produce nearly 70% greater resolution.

•At higher flow rates, the advantages of SPP-CSPs further increase.

Vancomycin

Teicoplanin

Teicoplanin aglycone

Sub 2 micron Titan Chiral Stationary Phases

Examples of ultrafast chiral separations on UHPLC with

teicoplanin, vancomycin and teicoplanin aglycone bonded phases

(50 mm x 4.6 mm ID columns)

UHPLC Optimization For Extra Column Volume

Optimized instrument

Ultra low dispersion needle and seat

75 µm i.d. connection tubing

0.6 µL detector flow cell

Stock instrument

Stock injection needle and seat

170 µm i.d. connection tubing

1 µL detector flow cell

Tröger's base

Instrument Agilent 1290 Infinity UHPLC

Stationary

phase

Cyclofructan-7-DMP 2.7 µm SPP (5 x

0.46 cm)

Mobile

phase

70:30 heptane:ethanol

Flow rate 2.5 mL/min

Tcol ambient (22 °C)

Tubing Studies in SFC

All tubings were cut same length (11.5 cm from injector to column and 20.0 cm from column to detector

Tubing diameter (µm) Manufacturer and part number

50 SGE analytical science ( PN:0624253)

75 SGE analytical science (PN:0624294)

127 Sigma-Supelco (PN: Z227293)

254 Sigma-Supelco (PN: Z226661)

508 Sigma-Supelco (PN: Z227293)

• Column used for studies: 5 x 0.46 cm teicoplanin bonded to high efficiency titan particles

Tubing selected for the studies in SFC

minutes

N1= 3970 ± 19tr1= 23.5 s

tr2= 26.7 s

N1= 3650 ± 30tr1= 22.8 s

tr2= 25.9 s

N1= 3100 ± 44tr1= 21.4 s

tr2= 24.2 s

Column: 1.9 µm NPSD Teicoplanin Flow rate: 5 mL/min

5 cm x 0.46 cm MP: 90:10:0.1 CO2 :MeOH:TEA

3-Phenylphthalide

254 µm tubing

127 µm tubing

75 µm tubing

However this common strategy for reducing extra column band broadening doesn’t necessarily work for SFC!

Ramifications of the Choice of Connection Tubings Ultrafast SFC

B) Retention factors of the achiral andchiral probes as a function of tubing internal diameters.

A) Comparison of efficiencies for achiral (1,3 dinitrobenzene) and chiral probes (3-phenylphthalide) as a function of the tubing internal diameters.

Column: 5 x 0.46 cm i.d., teicoplanin bonded 1.9 μm NPSD silica. For chiral analyte: MP: 80:20 CO2: MeOH at 2.7 mL/min. For 1,3 DNB, 90:10 CO2:MeOH at 2.8 mL/min. Back pressure regulator was maintained at 10 MPa. Column temperature: ambient.

Sampling Frequency, Response Times and

Embedded Signal Filtration

in

Fast High Efficiency Liquid

Chromatography

The chromatography detector capabilities and settings

become crucial

Sampling frequency (or sample rate) = the number of samples per second

Therefore, 1 Hz is one sample per secondand 100 Hz is one hundred samples per second

In chromatography, the sampling frequency andthe peak width determine the “points per peak” orhow many points are used to construct the peak seen on the digital chromatogram.

What is meant by a detector’s response time?

Effect of detector sampling rate and response time on efficiency (N) and resolution (Rs) in ultrafast chromatographic separations. BINAM analyzed on CF7-DMP SPP (3 cm × 0.46 cm), MP = 90:10 heptane–ethanol, 4.0 mL/min, Tcol = 22 oC; 1 Hz = 1 s–1, Agilent 1290 Infinity UHPLC.

Comparison of digital filters in Jasco SFC for a chiral probe: (3-phenylphthalide).

• The efficiencies represent the moment analysis of exponentially modified Gaussians using PeakFit program.

• Column: 5 x 0.46 cm i.d., teicoplanin bonded 1.9 µm NPSD silica. • Column temperature: ambient.• MP: 90:10 CO2:MeOH at 5 mL/min.

Data sampling frequency: 100 Hz

“Sampling frequency, response times and embeddedSignal filtration in fast, high efficiency liquid chromatography:

A tutorial.” in: Analytica Chemica Acta 907 (2016) 31-44.

For modern UHPLC and SFC detector deficienciesand idiosyncrasies see:

Some quick points:1) 20 points per peak are inadequate and unsupported by theory.2) Sampling frequency and response times can affect peak shape.3) Sampling frequency and digital filtering can affect: tr, noise

amplitude, peak shape and width in a complex fashion.4) Most chromatographers do not understand these effects.5) Some recent publications on these topics are incorrect.

Frictional heating can cause disparate effects in the efficiency of ultra-fast HPLC separations. Things that must

be accounted for include:

1) longitudinal temperature differences that do not hurt efficiency,

2) radial temperature differences that do hurt efficiency,

3) different mobile phases have different viscosities, heat capacities, densities and thermal conductivities.

1) High flow rates (>300 bar) generate substantial axial temperature differentials. In our work these axial temperature differences ranged from 11 to 18 oC.

2) A first order approximation of the maximum radial temperature difference, ∆TR, that can develop is: ∆TR = u(dP/dz)R2

4λrad

Temperature Effects

Where u is the superficial flow velocity in m/s, dP/dz is the change in pressure in the direction of the column axis (z) per unit length in N/m3, R is the column radius in m, and λrad is the approximate thermal conductivity of the mobile phase in the radial direction in W/ moC.

(3-8 oC at >300 bar)

Reversed PhaseTeicoplanin SPP CSP

(Water/MeOH)

Patel, et al., Gone in Seconds: Praxis, Performance, and Peculiarities of Ultrafast Chiral Liquid Chromatography with Superficially Porous Particles.Anal. Chem. 87 (2015) 9137-9148. DOI: 10.1021/acs.analchem.5b00715

Thermostated at 25°C Ambient temperature 22°C

This is now routine for us and the main impediment for doing routine sub-second separations is instrumental

limitations.

How do we proceed to further improve?

Lekker Sub-Second Separations(Liquid Chromatography)

Anal. Chem. (2016) DOI: 10.1021/acs.analchem.6b02260

Instrument setup to minimize extra column band broadening

36

Source Current “ultrafast” conditions Further modification

Injector

Ultra-low dispersion needle seat and

needle (Agilent P/N 5067-5189)

Bypass auto sampler, Rheodyne manual

injectors with 1 µL loop sizes

Injector to column connection tubing

Default connection tubing were

replaced by 250 mm x 75 µm i.d.

nanoViper connectors (Thermo Fisher

scientific, MA)

250 mm x 75 µm i.d. nanoViper

connectors will be replaced with 70 mm

x 75 µm i.d. nanoViper connectors

or

Direct connect injector to column

Column to detector connection tubing

Column is directly connected to the

detector

Column is directly connected to the

detector

Detector flow cellDetector flow cell with volume of 1 µL Detector flow cell with volume of 0.6 µL

Instrument – Agilent 1290 UHPLC

37

Flow

(A) (C)(B)

Selected column design0.5 cm x 4.6 mm i.d

Direct connection of column to the detector

Direct connection of column to the detector and arrangement of Rheodyne manual injector

Instrument Setup

0 0,5 1 1,5 2

Ab

so

rba

nce

Time (Seconds)

0.93 s

Stationary phase: spp-Quinine, 5 mmx 4.6 mm (I.D.)Mobile phase ACN:20 mM ammonium formate=70:30 (v/v), 5 mL/minDetection: 254 nmmanual injection 1uL with 7520 Rheodyne, 7cm x 75 µm I.D. Nanoviper. 1.0 µL detector, G4212_60008,5 cm precolumn, UHPLC filter bypass.

0.54 s

Sampling frequency: 160 HzResponse time: 0.016 s

Enantiomeric SeparationsD,L- DNB-Leucine

HILIC Separation

39

A: mellitic acidB: 4-aminosalicylic acid

B: 0.89 s

Stationary phase: spp-silica, 5 mmx 4.6 mm (I.D.)Mobile phase: ACN:15mM ammonium acetate= 94:6 (v/v), 5 mL/minDetection: 220 nmmanual injection 1uL with 7520 Rheodyne, 7cm x 75 µm I.D. Nanoviper. 1.0 µL detector, G4212_60008, 5 cm precolumn, UHPLC filter bypass.

A: 0.49 s

0 0,5 1 1,5 2

Ab

so

rba

nce

Time (Seconds)

Sampling frequency: 160 HzResponse time: 0.016 s

40

-5000

0

5000

10000

15000

20000

25000

30000

0,0 0,5 1,0 1,5 2,0

Abso

rban

ce

Time (Seconds)

Methyl

benzenesulfonate

4-Formylbenzene-1,3-

disulfonic acid

5.0 mm x 4.6 m m i.d. 2.7 µm SPP Teicoplanin. Sampling frequency: 160 Hz, response time: 0.016 sMobile phase: ACN: 20 mM NH4CO2H (40:60), Flow rate: 5.0 mL/min, Detection: UV at 220 nm ( Chromatogram is shown with power transformation where power (n) is 2)

Selected Separations in the Reversed Phase Mode

Reversed Phase Separation of Amino Acids

Stationary phase: Titan- Teicoplanin, 5 mmx 4.6 mm (I.D.)Mobile phase ACN:H2O= 15:85 (v/v), 5 mL/minDetection: 220 nmmanual injection 1uL with 7520 Rheodyne, 7cm x 75 µm I.D. Nanoviper. 1.0 µL detector, G4212_60008,5 cm precolumn, UHPLC filter bypass.

0 0,5 1 1,5 2

Ab

so

rba

nce

Time (seconds)

A: 0.26 s

A: AspB: β-AlaC: Trp-methyl ester

B: 0.65 s

C: 0.99 s

Sampling frequency: 160 HzResponse time: 0.016 s

1.0 sec

A: Gly-AspB: Gly-Val

0 0,5 1 1,5 2

Ab

so

rba

nce

Time (Seconds)

B: 0.95 s

Stationary phase: SPP- Teicoplanin, 5 mmx 4.6 mm (I.D.)Mobile phase ACN:20 mM ammonium formate=26:74 (v/v), 5 mL/minDetection: 210 nmmanual injection 1uL with 7520 Rheodyne, 7cm x 75 µm I.D. Nanoviper. 1.0 µL detector, G4212_60008,5 cm precolumn, UHPLC filter bypass.

A: 0.68 s

Sampling frequency: 160 HzResponse time: 0.016 s

Single Amino Acid Polymorphism (SAAP)

Analyte Chromatographic Conditions(stationary phase, mobile phase & flow rate)

tR1

(seconds)

tR2(Seconds)

Chiral separations

1. DNPyr-DL-Leucine Teicoplanin, 60:40 (MeOH:20 mM NH4CO2H), 5 mL/min

0.56 0.91

2. DNPyr-DL-Norvaline Teicoplanin, 70:30 (MeOH:20 mM NH4CO2H), 5 mL/min

0.66 1.00

3. (±)-4-Methyl-5-phenyl-2-oxazolidinone

Teicoplanin, 100% MeOH

0.60 0.98

4. N-Acetyl-Alanine Teicoplanin, 40:20:40 (MeOH: ACN: 5 mM NH4CO2H), 4 mL/min

0.56 0.99

5. N-(3,5-Dinitrobenzoyl)-DL-Leucine

Quinine, 70:30 (ACN:20 mM NH4CO2H), 5 mL/min

0.56 0.92

Achiral separations - HILIC

6. Mellitic acid + Benzamide

Cyclofructan, 95:5 (ACN:15 mM NH4CH3CO2), 5 mL/min

0.49 0.90

7. Mellitic acid + Benzamide

Silica, 95:5 (ACN:15 mM NH4CH3CO2), 5 mL/min

0.48 0.91

8. Mellitic acid + 4-Amino salicylicacid

Silica, 94:6 (ACN:15 mM NH4CH3CO2), 5 mL/min

0.48 0.93

9. Mellitic acid + 2,3-dihydroxybenzoic acid + 4-Amino salicylicacid

Silica, 94:6 (ACN:15 mM NH4CH3CO2), 5 mL/min

0.48 0.66(tR3 –0.93)

10. 4-Formyl-benzene-1,3-disulfonic acid +N-Ac-D-Alanine +Methyl benzenesulfonate

Teicoplanin, 70:30 (ACN: Water), 5 mL/min

0.40 0.61(tR3 – 0.87)

Achiral separations - Reversed phase mode11. Acetylsalicylic acid +

SalicylamideTeicoplanin, 35:65 (ACN:20 mM NH4CO2H), 5 mL/min

0.60 0.94

12. Salicylicacid + Methylsalicylate

Teicoplanin, 40:60 (ACN:20 mM NH4CO2H), 5 mL/min

0.61 0.93

13. 4-Formyl-benzene-1,3-disulfonic acid +Methyl benzenesulfonate

Teicoplanin, 40:60 (ACN:20 mM NH4CO2H), 5 mL/min

0.55 0.87

14. Dansyl-Asp + Gly

Teicoplanin, 30:70 (ACN:Water), 5 mL/min

0.44 0.81

15. Asp-Asp-Asp-Asp + Gly-Gly

Teicoplanin, 33:67 (ACN:20 mM NH4CO2H), 5 mL/min

0.47 0.88

16. Asp + β-Ala

Teicoplanin, 35:65 (ACN:Water), 5 mL/min

0.44 0.78

17. Gly-Asp + Gly-Val

Teicoplanin, 26:74 (ACN:20 mM NH4CO2H), 5 mL/min

0.59 0.84

18. Asp-Asp + Gly-Trp

Teicoplanin, 42:58 (ACN:20 mM NH4CO2H), 5 mL/min

0.56 0.98

19. Glu-Glu + Gly-Leu

Teicoplanin, 40:60 (ACN:20 mM NH4CO2H), 5 mL/min

0.52 0.90

20. Glu-Asp + Gly-βAla

Teicoplanin, 42:58 (ACN:20 mM NH4CO2H), 5 mL/min

0.54 0.99

Effect of sampling frequency with coupled noise removing Gaussian kernel embedded in the data acquisition software of Agilent’s UHPLC. A real sub-second separation of dansyl-L-aspartic acid and glycine (in order of elution) under one second at (A) 160 Hz, 0.016 s, (B) 40 Hz, 0.13 s, and (C) 10Hz, 0.5 s. Column - 0.5 cm x 4.6 mm i.d. SPP Teicoplanin, ACN: water (30:70), 5 mL/min, detection – UV at 220 nm.

20 points/peak?

Peak profile of a solute without the column

Conditions: Uracil, Mobile phase - 80/20 ACN/20 mM NH4FA, Flow rate 0.8 mL/ min, 254 nm.

Note that for short tubes and residence times, the Aris-Taylor Gaussian dispersion breaks down.

Tailing is characteristic of short tubings when there not enough residence time of the analyte in the tubes. This peak was fitted into the PeakFit v.12, and modelled as an Exponentially Modified Gaussian (EMG) with a R2 of 0.9904. The second moment of this peak is found to be 0.01156071. Similarly, the 0.5 x 0.46 cm i.d. column is inserted into the system and second peak moment is calculated by fitting the data to an Exponentially Modified Gaussian (EMG) with a R2 of 0.9990. The second moment is found to be 0.19160736.

Based on this information, the extra-column variance is: 100 x (0.01156071)/0.19160736 = 6.0% of the peak variance

At 5.0 ml/min, this variance is > 40%

250 milliseconds

Application of power transforms in sub-second chromatography of 3 components (mellitic acid, 2,3-dihydroxybenzoic acid, and 4-aminosalicylic acid). (A) The original sub-second chromatogram,(B) shows the deconvoluted chromatogram into three exponentially modified Gaussian peaks and (C) power transform with cubic of the original data. Column - 0.5 cm x 4.6 mm i.d. 2.7 µm SPP silica, Mobile phase- ACN:15 mM ammonium acetate= 94:6 (v/v), 5 mL/min at 220 nm.

The Effect of “Power Transform”

* Grushka, E. Anal. Chem. 1970, 42, 1142-1147.

Peak Capacity

For the 2nd dimension of LC x LC and expanding the separation time to 3 sec. & with N = 1200, peak

capacities of 20 or greater are possible.

Effect of sampling frequency on the efficiency and noise level in high efficiency

chromatography. System 3, time constant fixed at 0.01s. The first peak is uracil and

second peak is that of phenol. Injection volume 1 µL, mobile phase: 80:20 ACN:H2O at 1.8

mL/min, detection wavelength 254 nm.

Effect of the choice of the time constant on the efficiency and noise level. System 3 with a fixed sampling frequency of 100Hz. The first peak is uracil and second peak is that of phenol. Injection volume 1 µL, mobile phase: 80:20 ACN:H2O at 1.8 mL/min, detection wavelength 254 nm. Peak efficiency (average of 3 measurements) calculated by the exponentially modified Gaussian method to account for tailing.

Instrumental Idiosyncrasies Affecting the Performance ofUltrafast Chiral and Achiral SFC

Characteristics of SFC instrument manufacturers

SFC Instrument

Manufacturer

Type of Back

Pressure

regulator

Optical detector type Maximum

Sampling

rate

Response time

range

Type of

digital filter

Agilent 1260

Analytical SFC

Diaphragm

based

Multiple Wavelength

Detector (G1365C)

1260 Infinity Multiple

Wavelength Detector VL

(G1365D)

Agilent 1260 Infinity

Diode-Array Detector

(G4212B)

80 Hz

20 Hz

80 Hz

0.025-16 s

0.2 -16 s

0.031 -16 s

Gaussian

kernel

(not

disclosed by

Agilent)

Waters

(Investigator

SFC System)Needle based

2849 UV/Visible detector

2998 PDA detector

80 Hz

80 Hz

0-5.0 s

0-5.0 s

Hamming

filter

(Waters

manual)

Jasco (Semi-

preparative SFC

System)

Needle basedUV-2075

X-LC-UV 3070

100 Hz*

100 Hz

0.05- 3.0 s

0.03-0.3 s

Time

accumulation

RC

Digital filter

(Jasco

manual)

Shimazdu Diaphragm

basedSPD-M20 A 100 Hz 0-2.0 s

RC filter

(not

disclosed by

Shimadzu)

Enantiomeric separation on optimized Jasco andAgilent SFC systems

• Column: Teicoplanin bonded 1.9 μm NPSD silica.

• Back pressure regulatorwas maintained at 10 MPa.

• MP: 55:45 CO2:MeOH at 5 mL/min.

Effect of backpressure regulator settings

Effect of backpressure regulator setting on the

dead time, retention time and baseline noise in

ultrafast SFC. Toluene is used a model analyte

Fourier transform of the corresponding

chromatograms (after injection disturbance)

van Deemter curves of the first eluting enantiomer of 3-phenylphthalide.

Column: 5 x 0.46 cm i.d., teicoplanin bonded 1.9 µm NPSD silica.

MP: 80:20 CO2: MeOH.

Back pressure regulator was maintained at 10 MPa.

Column temperature: ambient.

Assessment of peak shapes on two detectors used in SFC (Jasco UV 2075, and XLC 3070 UV).

(A) Close-up of 1,3 dinitrobenzene peak and the corresponding time derivative on a real sampling frequency of 100 Hz on XLC 3070 UV (0.03 s response time)

(B) Simulated Gaussian peak at 100 Hz with its corresponding time derivative.

(C) Close-up of 1,3 dinitrobenzene on upsampled data at 100 Hz with its corresponding time derivative on UV 2075 detector (0.05 s response time)

(D) Simulated Gaussian peak

upsampled from 20 Hz (red

circles) to 100 Hz (blue

circles) by linear interpolation

(MATLAB 2010Ra) with its

corresponding time derivative.

Both detectors have the same

cell volume (4 µL).

Stationary phase: SPP-Cyclofructan 6, 5 mmx 4.6 mm (I.D.)Detection: 220 nmMobile phase: ACN:15mM ammonium acetate= 95:5 (v/v), 5 mL/minmanual injection 1uL with 7520 Rheodyne, 7cm x 75 µm I.D. Nanoviper. 1.0 µL detector, G4212_60008,5 cm precolumn, UHPLC filter bypass.

0 0,5 1 1,5 2

Ab

so

rba

nce

Time (Seconds)

B: 0.93 sA: 0.48 s

A: mellitic acidB: benzamide

Sampling frequency: 160 HzResponse time: 0.016 s

HILIC Separation