Technical Review

Chromatography, Vol.28 No.2 (2007)

Technical Review

High−Speed Analyses using Rapid Resolution Liquid Chromatography
on ZORBAX column packed 1.8 µm Particles
Tatsunari Yoshida(1), Ronald E. Majors(2) and Hiroki Kumagai(1)(1) Agilent Technologies, Inc. 9−1 Takakura−Cho, Hachioji−Shi Tokyo, 192−8501 Japan(2) Agilent Technologies, Inc 2850 Centerville Road Wilmington, Delaware 19808 USA

Abstract
The potential of high−speed analyses by Rapid Resolution Liquid Chromatography (RRLC) and RRLC/mass spectrometry (MS) on 1.8 µm
porous particles packed into short columns operated at high flow rate was investigated and compared to the performance of 5 µm porous particles packed into conventional columns. Using similar chemistries, the ease of conversion from conventional HPLC to RRLC method wasdemonstrated. In order to display the practicality of RRLC separations, the isocratic analysis of pesticides and the gradient analysis of catechins in Japanese Green Tea were selected. In isocratic analysis, compared to the conventional method, the time could be reduced by a factorof 31 at the highest flow rate. In gradient elution, the fastest separation was achieved by RRLC on 50 mm×4.6 mm i.d. or ×2.1 mm i.d. RRLC
column packed 1.8 µm particles. The analysis time was reduced by a factor of 15, compared to the conventional method.
Keywords: Rapid Resolution Liquid Chromatography, High throughput, Fast LC, sub−micron, 1.8 µm, pesticide, cathechinhigh−pressure liquid chromatography using a capillary column

1. Introduction
Over the years, separation resolution and reduction of analysis

packed 1−1.5 µm non−porous silica particles was introduced intime has continually improved in High Performance Liquid Chro-1997 [2]. Since then, HPLC using smaller particles has become

matography (HPLC). For further improvement, column efficiency

more popular.

must be increased. The relationship among separation efficiency,

In this paper, the technique of the use of small particles (sub−the mobile phase linear velocity and particle size was investigated

2 micron) packed into short columns run at a high flow rate hasin detail in the early 1970 s [1]. This and other systematic investi-

been termed Rapid Resolution Liquid Chromatography (RRLC).

gations have led to high−throughput and high−resolution HPLC

Recently, RRLC analysis has become a routine method in the phar-that we know today. The major advantages of high−throughput

maceutical industry. However, such a fast analysis technique is not

HPLC are the increase in throughput and the reduction in the

only for the pharmaceutical field. In this study, we apply to the fast

analysis cost. The shortening in analysis time is due to the use of a

RRLC technique to a more general field in the analysis of chemical

shorter column length. However, a shorter column may lead to acompounds. Today, the multi−residue screening method of pesti-

loss of theoretical plates, hence a decrease in chromatographic

cides established by the Japanese Food Hygiene Law method [3, 4]

resolution that may be required for a complex mixture of com-

use a 250 mm×4.6 mm i.d. column packed with 5 µm octadecyl (Cpounds. To offset the potential loss of resolution, the use of smaller18) silica gel used in the isocratic elution mode. These columns

size particles has resulted in more efficient columns. Further, ultra

typically produce an analysis time of 30 min. However, with new

Correspondence:(1) Agilent Technologies, Inc. 9−1 Takakura−Cho, Hachioji−Shi Tokyo, 192−8501 Japan(2) Agilent Technologies, Inc 2850 Centerville Road Wilmington, Delaware 19808 USA
Tel：0462−60−9925
Fax：0462−60−8676
E−mail: tatsunari_yoshida@agilent.com―８１―

Chromatography, Vol.28 No.2 (2007)

ZORBAX columns with 600 bar upper pressure limit were

RRLC technology, analysis time can be significantly reduced with-purchased from Agilent Technologies (Santa Clara, CA, USA).

out losing chromatographic resolution. In addition, gradient elution

HPLC grade water was prepared by Milli−Q system (Milli-

RRLC has been applied for the analysis of nutritional components

pore, Yokohama, Japan).

(catechins) in green tea using the same approach.

2.3. Chromatographic conditions

In this study, we had two goals: 1) to demonstrate the ease of

2.3.1. RRLC/DAD separation using Isocratic elution

conversion of a traditional HPLC method to a RRLC method and2) to investigate the potential of RRLC and RRLC/MS on 1.8 µm

Pesticides standards were dissolved using acetone or MeOH

porous particles packed into short columns operating at a high flow

and diluted with MeOH. For Japanese Food Hygiene Law method,rate compared to the performance of 5 µm porous particles packed

the separation of phenyl urea pesticides was performed on ZOR-

conventional columns.

BAX Eclipse XDB−C18 (250 mm×4.6 mm i.d., 5 µm) analyticalcolumn using the DAD detector set at a wavelength of 250 nm. The

2. Experimental

flow rate was 0.7 mL/min with the column compartment tempera-

2.1. Apparatus

ture set at 40℃ and an injection volume of 20 µL. Solvent A andsolvent B were water and MeCN, respectively. The isocratic elu-

All analyses were performed on an Agilent 1200 Series Rapid

tion condition was A/B (3/7).

Resolution LC system (1200 RRLC system; Agilent Technologies,
Hachioji−shi 1−9, Tokyo, Japan) equipped with on−line degasser,

For RRLC/DAD method, the pesticide separation was per-high−pressure binary pump, autosampler with temperature−con-formed on a ZORBAX Eclipse XDB−C18 Rapid Resolution HT

trolled sample compartment, thermostated column compartment,(RRHT) (50 mm×4.6 mm i.d., 1.8 µm) analytical column with theand photodiode array detector (DAD). For data processing and ac-same detection conditions. The flow rate was 0.7 mL/min with aquisition, Chemstation 01.03 (Agilent Technologies) was used. The

column compartment temperature was set at 40℃ and an injection

LC/MS system included the 1200 RRLC / 1100 MSD − liquidvolume of 4 µL was used. The mobile phase was the same as

chromatograph / mass spectrometer, and the LC/MS system wasabove.

operated in an external injection mode. In general, conventional

2.3.2. RRLC/MS approach using Isocratic elution

DAD analysis with the 1200 RRLC system was performed with a

For RRLC/MS method, the pesticide separation was per-normal instrument plumbing configuration, which has an internal

formed ZORBAX Eclipse XDB−C18 RRHT (50 mm×2.1 mm i.d.,system volume of approximately 600−800 µL. Components in-

1.8 µm) analytical column. The flow rate was 1.3 mL/min with astalled included internal tubing connections with 0.17 mm i.d. cap-

column compartment temperature of 40℃ and an injection volumeillary tubing, a 400 µL static mixer, damper (pressure dependentof 0.1 µL. Solvent A and solvent B were 5 mM ammonium formate

volume ranging from 80 to 280 µL), and 10 mm path length DAD

in water and MeCN, respectively. The isocratic elution condition

flow cell (13 µL). For RRLC analysis using 2.1 mm i.d. column,

was A/B (3/7). The LC/MS ionization was performed by Electro−

studies were performed on a 1200 system with lowest delay vol-Spray Ionization (ESI). Typical operating conditions of the ESI in-ume configuration that had an internal volume of approximately

terface in negative mode were as follows: vaporized temperature,

120 µL. Components installed included internal tubing connections

350℃; nebulizer gas, nitrogen, at a of pressure of 50 psi; drying

with 0.12 mm i.d. capillary tube and a 6 mm path length DAD flow

gas, also nitrogen, at a flow rate 10 L/min and capillary voltage,cell (5 or 1.7 µL) and without the static mixer and damper.

−3500 V. ESI in negative mode produces the [M−H]− molecular

2.2. Reagentsadduct ion. Quantitative analyses was performed by selected ion

HPLC grade acetonitrile (MeCN) and methanol (MeOH), pes-monitoring (SIM) mode. The monitoring m/z were as follows: 1)

ticides standards (Diflubenzuron, Tebufenozide, Hexaflumuron,

Diflubenzuron (m/z=309); 2) Tebufenozide (m/z=351); 3) Hexaflu-

Teflubenzuron, Lufebenzuron, Flufenoxuron, and Chlorluazuron),muron (m/z=459); 4) Teflubenzuron (m/z=379); 5) Lufebenzuron

catechin standards (Gallic acid, Gallocatechin, Epigallocatechin,(m/z=509); 6) Flufenoxuron (m/z=487); and 7) Chlorluazuron (m/zCatechin, Caffeine, Epicatechin, Epigallocatechin gallate, Gallo-=540).

catechin gallate, Epicatechin gallate, and Catechin gallate), alk-

2.3.3. RRLC/DAD separation using Gradient elution

lyphenons, (acetanilide, acetophenon, propiophenon, butyrophe-

Catechin standards for identification were dissolved in and di-non, benzophenon, valerophenon, hexanophenon, heptanophenon,

luted with HPLC grade water. The Japanese green tea sample wasand octanophenon) were purchased from Wako Pure Chemical In-

diluted four fold using HPLC grade water.

dustries, Co., Ltd (Osaka, Japan). All other reagents used were of

The separation of the Japanese green tea sample was per-the highest grade commercially available. Japanese Green Tea wasformed on three different ZORBAX SB−C18 (150 mm×4.6 mm i.

also purchased commercially at a local supermarket.

d., 5 µm), (75 mm×4.6 mm i.d., 3.5 µm) and SB−C18 RRHT (50―８２―Chromatography, Vol.28 No.2 (2007)

Tatsunari Yoshida, Ronald E. Major and Hiroki Kumagai

mm×4.6 mm i.d., 1.8 µm) analytical columns and with a DAD
wavelength setting of 210 nm. The flow−rate was 1.0 or 5.0 mL/
min with a column compartment temperature of 40℃ and an injection volume of 5 µL. Solvent A and solvent B were 0.1 % phosphoric acid in water and MeCN, respectively. For the 150 mm×4.6
mm i.d. column, the gradient elution condition was: initial concentration of 10 % B with programming to 15 % B over 7.5 min, andto 27 % B over 7.5 min. Then, the column was equilibrated under
the initial condition for 10 min prior to next injection. For 75 mm×4.6 mm i.d. size column, the gradient condition was 10−15 %B in
3.75 min, 15−27 %B in 3.75 min, and 5 min re−equilibration timeat the initial condition. For 50 mm×4.6 mm i.d. size column, thecondition was 10−15 %B in 2.5 min, 15−27 %B in 2.5 min, and 3
min re−equilibration time. For the high−throughput analysis at 5

Figure 1. The simultaneous separation of the phenyl urea pesticides. Shown are separations of phenyl urea pesticides
standard on (a)ZORBAX XDB−C18 (250 mm×4.6 mm
i.d., 5 µm) and (b) ZORBAX Eclipse XDB−C18 RRHT
(50 mm×4.6 mm i.d., 1.8 µm) analytical column. The
Peaks, in order of elution, are 1= Diflubenzuron, 2=Tebufenozide, 3=Hexaflumuron, 4=Teflubenzuron, 5=
Lufebenzuron, 6=Flufenoxuron, and 7=Chlorluazuron.
Detail conditions are shown in text.

mL/min flow rate using 50 mm×4.6 mm i.d. column, the conditionwas 10−15 %B in 0.5 min, 15−27 %B in 0.5 min, and 0.6 min re−equilibration time. For 50 mm×2.1 mm i.d. column at 0.21 or 1.05
mL/min, the gradient segments were the same conditions as that for
the 50 mm×4.6 mm i.d. column.
2.3.4. RRLC/MS approach using Gradient elution
For RRLC/MS method, the catechins separation was performed on a ZORBAX SB−C18 RRHT (50 mm×2.1 mm i.d., 1.8µm) analytical column. The flow−rate was 1.05 mL/min with a col-over 0.7 min and an isocratic hold for 0.2 min. The column wasumn compartment temperature of 40℃ and an injection volume ofthen equilibrated at the initial condition for 0.4 min prior to next in-

0.5 µL. Solvent A and solvent B were 0.2 % formic in both waterjection.

and MeCN, respectively.
The ESI conditions were as follows: vaporized temperature,

3. Results and discussion

350℃; nebulizer gas, nitrogen, at a of pressure of 50 psi; drying

When converting a conventional HPLC method to a fast or ul-gas, also nitrogen, at a flow rate 10 L/min and capillary voltage,

trafast HPLC method, the following parameters may require opti-−3500 V of negative and 3500 V of positive. ESI of catechins inmization: analysis time, gradient time and slope, flow rate and in-−negative mode produces the [M−H] molecular adduct ion in thejection volume. The detail method transfer consideration was de-Caffeine in positive mode produces the [M+H]+ molecular adductscribed in Ref [5, 6].

ion. The monitoring m/z were as follows: 1) Gallic acid, m/z=169;

3.1. RRLC Separation

2) Gallocatechin and 3) Epigallocatechin, m/z=305; 5) Caffeine

3.1.1. RRLC/DAD separation using Isocratic elution

m/z=195; 5) Catechin and 6) Epicatechin, m/z=289; 7) Epigallo-

These pesticides are widely used in agriculture for a large

catechin gallate and 8) Gallocatechin gallate, m/z=457; 9) Epicate-number of crops. The detection of their residues in food has caused

chin gallate and 10) Catechin gallate, m/z=441.

a great deal public concern. The multi−residue screening method of

2.3.5. Analytical precision under the fastest RRLC condition

these phenyl urea pesticides has been established by Japanese Food
Hygiene Law method [3, 4]. In this conventional method, an

Alkylphenone standards were dissolved using acetone or

isocratic separation has been developed using a 250 mm×4.6 mm i.

MeOH and diluted with MeOH.
The analytical precision of the alkylphenones was tested ond. column packed 5 µm octadecyl silica gel and the MeCN/H2O (7/ZORBAX SB−C18 RRHT (50 mm×2.1 mm i.d., 1.8 µm) analyti-3) solution as a mobile phase. These phenyl urea pesticides havecal column and on the DAD wavelength setting of 245 nm. The

the positive charge in acidic aqueous solution because they haveflow rate was 1.3 mL/min with a column compartment temperatureamine groups in the each compound. Therefore, these compoundsof 60℃ and an injection volume of 1 µL. Solvent A and solvent B

could interact with the surface silanols on silica gel and cause thewere 0.1 % triflouroacetic acid (TFA) in water and 0.1 % TFA inpeak tailing. The ZORBAX Eclipse XDB−C18 column (250 mm×

MeCN, respectively. The gradient elution condition was as fol-

4.6 mm i.d., 5 µm), thoroughly endcapped to cover active silanols,

lows: initial concentration 35 %B with programming to 95 %B

was used for the separation of these pesticides. Fig.1 (a) shows the―８３―Chromatography, Vol.28 No.2 (2007)

Figure 2. The ultrafast RRLC/MS of the phenyl urea pesticide
standard.
The phenyl urea pesticides standard was separated using
ZORBAX Eclipse XDB−C18 RRHT(50 mm×4.6 mm i.
d., 1.8 µm) analytical column at 1.3 mL/min. The top
chromatogram is the negative total ion chromatogram(TIC). The other chromatograms are obtained by thenegative SIM mode. The peaks, in order of elution, are
1= Diflubenzuron (m/z=309), 2=Tebufenozide (m/z=351), 3=Hexaflumuron (m/z=459), 4=Teflubenzuron (m/z=379), 5=Lufebenzuron (m/z=509), 6=Flufenoxuron(m/z=487), and 7=Chlorluazuron (m/z=540). Detailconditions are shown in text.

Figure 3. HETP versus linear velocity for small particle sphericalbonded silica column [5].
Shown is a series of van Deemter curves for (a)5, (b)3.5
and (c)1.8 µm bonded spherical bonded silica column.
The column (a), (b) and (c) is ZORBAX XDB−C18 (30mm×4.6 mm i.d., 5 µm), ZORBAX XDB−C18 (30 mm
×4.6 mm i.d., 3.5 µm) and ZORBAX XDB−C18 RRHT
(30 mm×4.6 mm i.d., 1.8 µm) analytical column, respectively. The solute is octanophenone and the mobile
phase is 85:15 MeCN/water. The flow rate range is 0.05
mL (1.8 mm/sec) − 5.0 mL/min (18 mm/sec); temperature: 20℃.

25 min chromatogram of the seven standard phenyl urea pesticides.

RRLC/MS method is 31 times faster than that by the method of

Fig.1 (b) shows the 5 min chromatogram for the separation ofJapanese Food Hygiene Law method. RRLC coupled with MS

the seven standard phenyl urea pesticide using the 50 mm×4.6 mm

showed excellent experimental results in term of resolution, selec-

i.d. ZORBAX Eclipse XDB−C18 RRHT column packed with 1.8

tivity and sensitivity.

µm particle size octadecyl silica gel. This column and demonstrates

In a series of isocratic experiments, it is concluded that thethe same chemistry as the ZORBAX 3.5 µm and 5 µm bonded sil-

RRLC method could achieve a faster separation using the shorter

ica particles. As a result, both columns packed with 5 and 1.8 µm

column packed with the 1.8 µm particles at a high flow rate with-particles provided identical selectivity. RRLC method in isocratic

out loss in resolution.
The reason that the rapid, high−resolution separation is main-

elution could easily replace the conventional regulated method.

tained at a high flow rate is evident from van Deemter analysis.

3.1.2. RRLC/MS approach using Isocratic elution
The RRLC/MS separation of same pesticide mixture was at-

The separation efficiency H (height equivalent to theoretical plate,tempted and the chromatogram of standard sample is shown inHETP) in micrometers as a function of mobile phase velocity is de-Fig.3. In this case, a narrow bore ZORBAX Eclipse XDB−C18

scribed by the van Deemter equation, shown simplistically in

RRHT (50 mm×2.1 mm i.d., 1.8 µm) column was used. In this ex-

Equation 1.

periment, the flow rate in this experiment was reduced to 1.3 mL/

H=A+B/µ +Cµmin (23.4 mm/sec) for a resultant column backpressure of 550 bar.

(1)

The linear velocity (23.4 mm/sec) on 2.1 mm i.d. column would

where A, B, and C are constants and µ is the mobile phase linear

correspond to the 8 mL/min on the 4.6 mm i.d. column. The same

velocity (proportional to flow rate), measured in centimeters per

seven phenyl urea pesticides could be successfully separated using

second. The A term is a measure of the packing efficiency and is a

this narrow bore column at this high flow rate. In this case, the total

function of packing efficiency and particle size. The B term is aanalysis time by RRLC/MS method was only 0.7 min on the ZOR-

function of longitudinal diffusion, or diffusion in the mobile phase,

BAX Eclipse XDB−C18 RRHT (50 mm×2.1 mm i.d, 1.8 µm) col-and the C term is a function of the mass transfer between the sta-

umn without a loss in resolution. Furthermore, this analysis time bytionary and mobile phase as well as within the mobile phase.

―８４―Chromatography, Vol.28 No.2 (2007)

Tatsunari Yoshida, Ronald E. Major and Hiroki Kumagai

Figure 4. The separation of catechins in the Japanese Green tea
on the three columns.
The chatechins in the Japanese Green tea were performed using (a) ZORBAX SB−C18 (150 mm×4.6 mm
i.d., 5 µm), (b) ZORBAX SB−C18 (75 mm×4.6 mm i.
d., 3,5 µm), and (c) ZORBAX SB−C18 RRHT (50 mm
×4.6 mm i.d., 1.8 µm) column at 1.0 mL/min. The chromatogram (d) are obtained on ZORBAX SB−C18 (50mm×4.6 mm i.d., 1.8 µm) column at 5.0 mL/min. The
chromatogram (e) is enlarged in a separation window.
The Peaks, in order of elution, are 1=Gallic acid, 2=
Gallocatechin, 3=Epigallocatechin, 4=Catechin, 5=Caffeine, 6=Epicatechin, 7=Epigallocatechin gallate, 8=
Gallocatechin gallate, 9=Epicatechin gallate, and 10=
Catechin gallate.

Figure 5. The ultrafast RRLC/MS of catechins in the JapaneseGreen tea.
The catechins in the Japanese Green tea were separated
on ZORBAX SB−C18 RRHT (50 mm×2.1 mm i.d., 1.8µm) column at 1.05 mL/min. The chromatogram (a) is
the negative TIC. The chromatogram (b), (c), (d), (e),(f) and (g) is obtained by the SIM mode, (b) m/z=169,(c) m/z=305, (d) m/z=195, (e) m/z=289, (f) m/z=457
and (g) m/z=441. The peaks, in order of elution, are 1=
Gallic acid, 2=Gallocatechin, 3=Epigallocatechin, 4=
Catechin, 5=Caffeine, 6=Epicatechin, 7=Epigallocatechin gallate, 8=Gallocatechin gallate, 9=Epicatechingallate, and 10=Catechin gallate.

Within the C term, there is also a proportional dependency of the

togram obtained using a ZORBAX SB−C18 (75 mm × 4.6 mm i.

particle diameter squared. Another fallout of the decrease in parti-d.) column packed with 3.5 µm particles. If the internal column di-cle size is that the van Deemter curves tend to flatten out at higher

ameter among three columns is the same, the gradient time is pro-linear velocities and the minimum shifts toward the right. Fig. 3

portional to the column. In this case, as the column length isshows a series of van Deemter curves for 5, 3.5 and 1.8 µm bonded

halved, each gradient time should be half (i.e. 1st segment, B 10 %spherical silica columns. Researchers can easily see that the col-(5 min) to B 10 %(2.5 min) and 2nd segment, B 27%(15 min) to B

umn packed with 1.8 µm particles gives a flatter curve at high lin-

27 %(7.5 min)). Even switching to a 75 mm column reduced theear velocity than the 5 µm column [5]. Thus, these column can betotal time to just over 7.5 min (Fig. 4(b)). Fig. 4(c) shows the chro-run at faster flow rates (linear velocities) and the chromatographic

matogram separated using a ZORBAX SB−C18 RRHT (50 mm ×peaks maintain their efficiency yet the separation time decreases

4.6 mm i.d.) column packed with 1.8 µm particles. The same sepa-

proportional to the increase in flow rate.

ration could be achieved on the column that was only 50 mm in

3.1.3. RRLC/DAD separation using Gradient elution

length but in a third of the time − less than 5 min. The flow rate
among Figs. 4(a)−(c) was 1 mL/min (3.6 mm/sec). Using the

A comparative study of the chromatography of catechins inshorter column length can save the analysis time and solvent.

Japanese green tea on three different columns packed with 5, 3.5,
and 1.8 µm reversed phase C18 bonded material is depicted in Fig.

Next, an ultra fast separation at a high flow rate (5 mL/min =4. The theoretical plate number among the three columns is almost

18 mm/sec) was attempted. As mentioned above and indicated insame. The results for the ZORBAX SB−C18 (150 mm × 4.6 mm i.

Fig.3, the column packed with 1.8 µm particles has a flatter curved.) column packed with 5 µm particles (Fig. 4(a)) are typical forat high linear velocity, so peaks maintain their efficiency yet thethis standard column that is routinely used in many HPLC labora-

separation time decreases proportional to the increase in flow rate.

tories. On this conventional column, the catechins could be com-

The result of this approach is shown in Fig. 4(d). The total analysis

pletely separated as shown in Fig. 4(a). This gradient method re-time was less than 1 min. A nearly five fold faster separation speed

quired a total of 15 min analysis time. Fig. 4(b) shows the chroma-

could be achieved and the resolution of peaks maintained compara-―８５―Chromatography, Vol.28 No.2 (2007)

Table 1. Solvent consumption and column pressure drop

sumption as well. For the inner diameter of 2.1 mm i.d., the short-

Solvent A, B,
consumption[mL]

Analysis
time
[min]

Columndrop pressure[bar]

SB 5 µm
150 mm×4.6 mm i.d.
40℃, 1.0 mL/min

A;21.5
B;3.5
Total;25.0

15

56 barSB 3.5 µm
75 mm×4.6 mm i.d.
40℃, 1.0 mL/min

A;10.7B;1.8
Total;12.5

7.5

SB 1.8 µm
50 mm×4.6 mm i.d.
40℃, 1.0 mL/min

A;6.9
B;1.1
Total;8.0

5

SB 1.8 µm
50 mm×4.6 mm i.d.
40℃, 5.0 mL/min

A;6.9
B;1.1
Total;8.0

1

SB 1.8 µm
50 mm×2.1 mm i.d.
40℃, 0.21 mL/min

A;4.5
B;0.9
Total;5.1

5

SB 1.8 µm
50 mm×2.1 mm i.d.
40℃, 1.05 mL/min

A;4.5
B;0.9
Total;5.1

1

Columnest analysis time (below 1 min) was performed with ZORBAX SB−C18 RRTH (50 mm×2.1 mm i.d., 1.8 µm) column at flow rate
1.05 mL/min (18 mm/sec) with total solvent consumption 5.1 mL.
The column pressure (485 bar) was not as high as observed withZORBAX SB−C18 (50 mm×2.1 mm i.d., 1.8 µm) at highest flowrate 5.0 mL/min (18 mm/sec). It is always best to run the HPLC
column at the lowest pressure drop as possible. Column pressure

72 barcan be lowered by the use of shorter columns, lower flow rates, lowviscosity mobile phase and higher temperatures.
3.1.4. RRLC/MS approach for Gradient elution

95 barIn the above experiments using DAD, orthophosphoric acidwas used as modifier because of its low absorbance in the UV region of the spectrum, as low as 190 nm. However, this acid is un-

500 barsuitable for use in MS. A volatile mobile phase must be used forMS. Formic acid is frequently used as a modifier for LC/MS work.
88 barAlthough the separation selectivity may be altered by changing themodifier, there are no clear method conversion protocols with achange in the modifier, and thus the suitable separation conditions

485 barmust be determined by trial and error. After the optimum of gradient elution profile and mobile phase were determined, the catechins
could be completely separated on a ZORBAX SB−C18 RRHT (50mm × 2.1 mm i.d.) column packed with 1.8 µm particles. The MS

ble to the separation at 1 mL/min. The ultimate separation speed

chromatogram is shown in Fig. 5. The total analysis time was 1.6

was obtained using the shortest column packed with the small par-min at 1.05 mL/min (18 mm/sec). This LC/MS separation wasticles (1.8 µm) at the highest flow rate without a loss in resolution.

longer than the LC/DAD separation due to the change in selectivity

With respect to gradient separation, system volume (alsocaused by the modifier. Using the MS selectivity and MS parame-called the gradient delay volume or the dwell volume) must be

ter adjustment, the resolution of separation could be improved. In

noted. The gradient delay volume is the volume from the point of

this MS experiment, the sampling rate was adjusted to 0.08 min

mixing of the binary eluents to the column entrance. The lower(corresponding to peak half width). The sampling rate is also im-

flow rates used with smaller−diameter columns will increase theportant in MS detection since sometimes a fast acquisition rate cangradient delay time because the time needed to traverse the gradi-cause a cross talk phenomena.

ent delay volume is long. When the gradient delay time is large, the3.1.5 Analysis precision under fastest RRLC condition

gradient time should be changed in order to maintain resolution.

The repeatability of the gradient analytical run was tested forFurther, gradient delay volume will also affect regeneration time.

retention time, peak height and area using the mixture of nine al-

Larger delay volume needs a much longer regeneration time.

kylphenones. The narrow bore ZORBAX SB−C18 RRHT (50 mm

Therefore, the HPLC apparatus with the minimum gradient delay

×2.1 mm i.d., 1.8 µm) analytical column was used at 1.3 mL/min

volume is required for low flow rate separations [7].

(23.4 mm/sec). This linear velocity (23.4 mm/sec) at 2.1 mm i.d.

The solvent consumption for the chromatograms shown in

was corresponding to the 8 mL/min at 4.6 mm i.d.. At this flow

Fig. 4 was calculated, and the results are summarized in Table 1.

rate, the column compartment temperature was adjusted to 60℃

Furthermore, the pressure drops under the experimental conditions

and, therefore, the pressure drop was only 500 bar. The overlay re-were also listed in Table 1. The shorter the column, the proportion-sult of six replicate injections of alkylphenones is shown in Fig. 6.

ally lower is solvent consumption when columns are run at the

The deviation between individual chromatograms was quite small.

same flow rate. For the inner diameter of 4.6 mm i.d., the shortest

In the Table 2, the %RSD of retention time, peak height and area

analysis time (below 1 min) was obtained with ZORBAX SB−C18

are listed. The mean retention time precision is 0.2 % RSD. The

RRHT (50 mm×4.6 mm i.d., 1.8 µm) column at flow−rate 5.0 mL/mean peak height and area precision are 0.61 % and 0.47 % RSD,min (18 mm/sec) with total solvent consumption 8.0 mL. The

respectively. At this high flow rate and operating pressure, the re-smaller is inner diameter of column, the lower is the solvent con-producibility was quite satisfactory for all test compounds.

―８６―

Chromatography, Vol.28 No.2 (2007)

Tatsunari Yoshida, Ronald E. Major and Hiroki Kumagai

Table 2. Relative Standard Deviation (RSD)s (n=6) of the retention times, peak heights and peak areas.

Figure 6. Repetitive ultrafirst separations of alkylphenones (sixreplicate injections).
The chromatogram of the mixture of 9 kinds alkylphenones was obtained on ZORBAX SB−C18
RRHT(50 mm×4.6 mm i.d., 1.8 µm) analytical column.
The Peaks, in order of elution, are 1=acetanilide, 2=acetophenone, 3=propiophenone, 4=butyrophenone, 5=benzophenone, 6=valerophenone, 7=hexanophenone, 8=heptanophenone, and 9=octanophenone. See text forother chromatographic conditions.

No. Alkylphenons

Retention time
%STD

Height
%STD

Area
%STD

1 Acetanilide
2 Acetophenone
3 Propiophenone
4 Butyrophenone
5 Benzophenone
6 Valerophenone
7 Hexanophenone
8 Heptanophenone
9 Octanophenone
Average

0.64
0.40
0.13
0.16
0.16
0.09
0.08
0.07
0.08
0.20

0.45
0.88
0.26
0.79
0.60
0.59
0.57
0.83
0.53
0.61

0.69
0.74
0.27
0.35
0.38
0.73
0.36
0.23
0.46
0.47

min, a factor of 15. From the point of view of increasing sample
throughput, lowering solvent consumption and in time savings, it
was concluded that the fast RRLC technique is useful for not only
pharmaceuticals but also for chemical compounds currently analyzed by conventional HPLC on 250 mm×4.6 mm i.d. columns.
In the future, the fast RRLC technique will be used more
widely in other HPLC fields.

4. Conclusions
In this study, we applied the fast RRLC technique to theanalysis of general chemical compounds. Most applications ofReferences

these small particle columns have been in the area of pharmaceuti-[1]

R.E. Majors, J. Chromatogr. Sci. 1973, 11, 88−95.

cal analysis. Using a 1200 RRLC system equipped with higher ac-[2]

J.E. McNair, K.C. Lewis, and J.W. Joregenson, Anal. Chem.
1997, 69, 983−989.

quisition rate of detector, low dead volume system configurationand combined with a high−pressure HPLC system with 600 bar[3]pressure capability, the ultrafast RRLC analysis could be done with

[4]

Japanese Food Hygiene Law No.216 (1999)
R. Higuchi, Y. Tsuchida and H. Sakai, Niigataken Hoken
Kankyokagaku Hokensho Nenpo 2002, 17, 76−82.

satisfactory analytical precision.
[5]

RRLC separations could be easily achieved from many tradi-

T. Yoshida and R.E. Majors, Journal of Separation Science,
2006, 29, 2421−2432.

tional HPLC methods. In an isocratic example, one cycle analysis[6]time by traditional HPLC/DAD method that required 25 min could

M. Frank, “Achieving fastest analyses with Agilent 1200 Se-

be shortened to a RRLC/MS method to 0.8 min, a factor of 31. The

ries Rapid Resolution LC System and 2.1−mm Columns”,

same method transfer approach was carried out in a gradient sepa-

Agilent Technologies Application Note Publication Number

ration. One cycle analysis time by conventional HPLC/DAD

2006, 5989−4502 EN, March.
[7]

method of 15 min was shortened to a RRLC/DAD method of 1.0―８
７―

R.E. Majors, LCGC 21, 2003, 1124−1133.