LC Technical Note

How to Use Preparative HPLC - Part 2
Scaling up from Analytical HPLC

LT134
GL Sciences Inc.

It is not easy to find out optimal condition for preparative HPLC. Not only large volume of solvent but also
substantial amount of precious sample may be required for the evaluation of separation conditions, particularly inpreparative HPLC. Consequently, we recommend that the evaluation should be carried out using analytical column(4.6 mm I.D.) in the beginning. Condition for preparative HPLC can be investigated efficiently by using analyticalcolumn packed with the same gel as in preparative HPLC column.
In this note, Inertsil ODS-3 was taken as an example, and how to scaling up from analytical column to preparativecolumn is described.
(K. Kanno)

Conditions of preparative HPLC are generally optimized as the followingflowchart

Find out appropriate
separation conditions using
analytical column• Composition of mobile phase• Gradient programetc.

Evaluate loading amountusing analytical column

Described in LC Technical
Note No. 133.

Scaling up to preparativecolumn

Described in this note

Preparative HPLC
＜What is important to scale up＞
It is important to calculate ratio of cross-sectional area of preparative column to that of analytical column. The ratio
can be used as follows;

1)Increase flow rate in proportion to cross-sectional area of column
2)Increase loading amount (injection volume) in proportion to cross-sectional area of column

Innerdiameter
Cross-sectional area

4.6 mm

10 mm

14 mm

20 mm

30 mm

16.6 mm278.5 mm2

154 mm2

314 mm2

707 mm2

Ratio of the area※１

1

4.7

9.3

18.9

42.5

※1 Ratio of cross-sectional to that of 4.6-mm I.D. column was calculated.

GL Sciences LC Technical Note1) Increase flow rate in proportion to cross-sectional area ofcolumn
The figure shown right indicates relation between linear velocity of mobile
phase and height equivalent to theoretical plate (HETP) obtained with threecolumns packed with 10 μm particles.
Optimum flow rate, at which the lowest HETP is obtained, is 3.0 cm/min(0.5 mm/sec) for all the columns. Therefore, it can be said that flow rateshould be changed to maintain optimum linear velocity of 3.0 cm/min incase of scaling up from 10 μm particle packed analytical column to 10 μm
particle packed reparative one. It is important that particle size of the twocolumns is same because optimum flow rate changes also depending on
particle size.

2) Increase loading amount (injection volume) in proportion to cross-sectional area of column
The figure shown below indicates relation between loading amount and number of theoretical plates (N). Threecolumns with different inner diameters were used and compared. For example, maximum loading amount for eachcolumn at which N above 2000 can be obtained is follows;

6 mm I.D. approx. 1 mg

20 mm I.D.

approx. 10 mg

50 mm I.D.

approx. 70 mg

Number of theoretical plates (N)Since the maximum loading amount is proportional to cross-sectional area of column, it can be said thatsimilar separation should be achieved with preparative column as with analytical column by increasing
injection volume in proportion to cross-sectional area.

Loading amountGL Sciences LC Technical Note
＜Parameters to be changed for scaling up＞
In case of scaling up from a 4.6 mm I.D. analytical column to a 20 mm I.D. preparative column, cross- sectional
area of the column is approximately 19 times enlarged. Therefore, scaling up can be achieved by increasing flowrate and loading amount (injection volume) 19 times. Red letters represent parameters to be changed for scalingup.
ColumnEluentFlow rate
Column Temp.
Detection

Injection Vol.*: Inertsil ODS-3(10 μm, 4.6 mm I.D. ×250 mm): A) CH3CNB) H2O
A/B = 40/60, v/v: 500 µL/min: 40 ℃: UV 270 nm: 500 µL

ColumnScale up: Inertsil ODS-3(10 μm, 20 mm I.D. ×250 mm): A) CH3CNB) H2O
A/B = 40/60, v/v: 9.5 mL/min(9500 µL/min): 40 ℃: UV 270 nm: 9.5 mL (9500 µL)

EluentFlow rate

Increase flow rate andinjection volume 19 times.
Keep other parameters
unchanged

Column Temp.
Detection
Injection Vol.** Concentration of the sample solution is same＜An example of scaling up＞
Chromatograms before and after scaling up are shown below
HPLC conditions
ColumnColumn I.D.
EluentFlow rate
Column Temp.
Detection

Sample1: Inertsil ODS-3(5 m, 250 mm length): Discribed in each chromatogram: A) CH3CNB) 0.1 % TFA
A/B = 40/60, v/v: Discribed in each chromatogram: 40 ℃: UV 270 nm

Column I.D.
Flow rateInjection Vol.

: 4.6 mm
: 1.0 mL/min: 160 μL
Loading amount : 0.32 mg: Commercially available licorice extract (2.0mg/mL)
0246810Time (min)121416

Loading amount was 18.9 times increased.
1Column I.D. Flow rate Injection Vol.

: 20 mm: 18.9 mL/min: 3.0 mL
Loading amount : 6.0 mg

1. Glycyrrhizic acid02468Time (min)10121416GL Sciences disclaims any and all responsibility for any injury or damage which may be caused by this data directly or indirectly. We reserve theright to amend this information or data at any time and without any prior announcement.

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