Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC)

2017/02/04
K. Shinoda

Apparatus Composition

Injector
DegasserPump

ODS (Octadecylsilane): C18Mixer

MeOO SiSiO

C18H 37

MeColumn

Eluent A(aq. TFA (0.1%))

Eluent B(MeCN)

UV
DetectorWaste

PC for Data Acquisition✓ two types of HPLC: “analytical HPLC (to analyze your sample)” and “preparative HPLC (to purify your sample)”
How to Use

Please confirm thatthe baseline
became stable!
Time[chart example][preparative HPLC]a) peak collectionb) characterization(MALDI-TOF-MS)c) purity check(analytical HPLC)Time*After the injection, please wash the injector by MeOH.

3) wash by eluent B again✓ 10 − 15 min[chart example]
UV Absorbance

UV Absorbance[chart example (initializing)]

2) inject your sample* and start the run

UV Absorbance1) wash by eluent B → initialize by eluent A✓ 10 − 15 min each

Please continue
until the baseline
becomes stable !

TimeReverse-Phase High-Performance Liquid Chromatography (RP-HPLC)

2017/02/04
K. Shinoda

Difference between Analytical HPLC and Preparative HPLC
✓ flow rate
○ analytical (150 × 4.6 mm): ca. 1 mL/min*○ semi-prep. (250 × 10 mm): ca. 3.0 mL/min*○ prep. (250 × 20 mm): ca. 10.0 mL/min*✓ maximum loading amount○ analytical: ca. 0.5 mg○ semi-prep.: ca. 10 mg○ prep.: ca. 50 mg*In all cases, the pressure should be less than 10 MPa.

✓ method[example of analytical method]yy [%] = (x − 2 [min])× 2.5 [%/min]

0100

MeCN (%)

MeCN (%)[example of preparative method]

When the target compoundis eluted at x min, …

100✓ maximum loading volume○ analytical: ca. 100 μL○ semi-prep.: ca. 1 mL
○ prep.: ca. 5 mLy + 20
yy − 20

more slow gradient

5*0 2x 42 52
Time (min)the target compound will
be eluted at ca. 40 min.

020 21 41 61 62 75
Time (min)*In preparative scale, 0% of MeCN is not recommended because the compound may be precipitated.

Tips for Analytical HPLCMeCN (%)✓ another example of analytical method with shorter analyzing time
100In order to let more people touse analytical HPLC, it isrecommended to use the methodwith short analyzing time.

00 3

15 20Time (min)✓ It is recommended to analyze your sample by reversephase TLC (ex. ODS) before injection.
Such compound should be
removed before injection
as it should damage theODS column.
ODS short column will be effective to remove it.

Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC)

2017/02/04
K. Shinoda

Several Strategies of Purification

You have to keep
in mind that you willlose your compound
to some extent.

✓ If you don’t want to lose yourcompound, it is recommended tocollect the whole peak.

UV Absorbance

UV Absorbance✓ If you want to obtain the single peak,
it is recommended to collect the upper
half of the peak.

✓ If you couldn’t purify your compoundin one time purification, it will be effective
to conduct the second purification.
[key point in the second purification]

10 − 15 sec.
1 fraction○ changing the type of the column(e.g. YMC-Triart → ODS-Pack AM)○ using slower gradient methodTimeTime
Tips for Preparative HPLCUV Absorbance✓ Please don’t load too much.
Too much loading
may result in such
chromatogram, butthese two peaks may
be derived from thesame compound…

✓ Before the purification, please filter your sample solution toremove particulates, which may damage the column.
✓ Generally speaking, the amount of your compound will be decreased
to less than 50% after the purification.
✓ As in the case of analytical HPLC, it is better to use the methodwith shorter time.

Time✓ It is recommended to dissolve your compound in solvents which are used as eluents (e.g. aq. TFA (0.1%) or MeCN).
○ Polar solvents with strong dissolving power (e.g. DMSO, DMF) may inhibit the capture of your compound to the column.

✓ We have two ways to collect peaks (manual and automatic (fraction collector)). Please choose whatever you’d like.

Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC)

2017/02/04
K. Shinoda

Tips for Eluent✓ other candidates for eluent A: aq. HCO2H (0.1%)✓ other candidates for eluent B: MeOH (stronger eluent than MeCN)✓ It is recommended to degas both eluents just after their preparation (air bubble may be generated in flow paths).

Tips for Injector✓ The injection volume of your sample should be less than the maximum of the loop volume; 80% will be the limit.
✓ When you want to inject much more volume than the limit above, you may follow the following procedure.
1) inject a part of your sample; 2) wait 10 – 20 min (keep flowing with the initialization condition); 3) inject again;4) repeat the sequence above (1 – 3) until the whole of your sample is injected
Tips for Column✓ other candidates for C18 column: C4, C8, etc.

✓ You may use the column oven to heat the column (40 ºC, 60 ºC, etc.).

✓ When a column is clogged, please follow the following procedure.
1) Please connect the column in the opposite direction and wash with strong eluent such as i-PrOH.
2) Wash should be continued for O/N or 1 day with a 10% flow rate of the usual flow rate.
Tips for UV detector✓ general UV wavelength for peptides: 230 nm✓ general UV wavelength for proteins: 280 nm✓ other candidates for UV wavelength:○ 214 nm (aromatic residues-poor peptides)○ 254 nm (aromatic residues-rich peptides)○ 301 nm (indole ring (Trp residue))✓ We also have fluorescence detector and refractive index detector.

✓ When air bubble is contaminated,the chart will become as follows.
If you see such chart,
please purge the system.
During the purge, pleasetake the column off so as
not to damage the column.