Protein Analysis by
Ultra High Perfomance Liquid Chromatography (UHPLC)
Using
Size Exclusion Chromatography (SEC) for Molecular Weight
Determination and Photo Diode Array (PDA) Detector for
Identification.
Advanced Lab on Analysis on Bio-Molecules
Weizmann Institute, 2013
Lab manual
By
Dr. Shulamit Levin, Waters (TC) Israel
__________________________________________________________________________________
Shula Levin
shula_levin@waters.com
Tel: 052-9402443
Protein Analysis by UHPLC Lab Manual 2013,
Advanced Lab on Analysis on Bio-Molecules, Weizmann Institute, 2013
Introduction: Size Exclusion Chromatography (SEC)
Size-exclusion chromatography (SEC) is a separation technique, whereby proteins are separated based on
the differences of their size when dissolved in aqueous buffers. The protein molecules migrate through a
chromatographic column packed with gel particles, through which the sample components need to
permeate, either fully, partially or none at all, in order to be resolved from each other in time. The
various sample components elute out of the column into a detector which reveals them as peaks in a plot
called “chromatogram”. The chromatogram shows the detector response as function of time.
Fig. 1:
Permeation of various size molecules through gel particles. The larger sizes remain outside and advance
faster than the smaller ones.
Over the years, SEC has been known by a number of other names, such as exclusion chromatography,
steric-exclusion chromatography, restricted-diffusion chromatography, liquid-exclusion chromatography,
gel-filtration chromatography, and gel-permeation chromatography. Since the separation is based strictly
on the size of the sample components in solution, there should be no interaction with the column packing,
(adsorption, partition, etc.), as in all other modes of liquid chromatography.
The differential migration of the macromolecules through the column allows the determination of their
molecular weights, because the elution time of each macromolecule is inversely proportional to the
logarithm of its molecular weight, i.e., the larger the molecule, the shorter is its elution time. Markers of
known molecular weights are injected, and their molecular weights are used to create a calibration curve,
then the unknown is injected and its molecular weight is calculated from this calibration curve.
Fig 2:
Example of (A) a typical chromatogram of the protein markers (B) a calibration curve of these
macromolecules, showing how the Elution Time (or volume) is inversely proportional to the log (MW) of
the macromolecules.
A
B
The unknown is then injected and its retention time is used to extract its molecular weight from the
calibration curve
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Fig 3:
Unknown proteins can be injected into the SEC column and the software automatically calculates their
molecular weights based on the calibration curve.
Identification and Quanfitation of molecules using Photodiode Array (PDA)
Detector
A UV/VIS detector, whether operated in a fixed wavelength or variable wavelength, produces the
chromatogram as a two-dimensional (2D) output. This output plots time on the x-axis and response,
absorbance units (AU) in this case, on the y-axis. The chromatogram is then analyzed by integrating the
peaks curves to get area, getting their retention time (RT) from the peak maximum to identify them, and
perform quantitative analysis, by comparing their area to those of samples whose concentrations are
known, i.e, standards.
A photodiode array (PDA) detector produces a three-dimensional (3D) output. Similar to the UV/VIS
detector output, the output from the PDA plots time on x-axis and absorbance on y-axis. The third
dimension is the z-axis which plots wavelength. Usually 3D data is used for identification by spectral
analysis and 2D data for quantitative analysis.
Fig 4:
Three dimensional plot of Chromatograms and Spectra:
YZ->UV Spectrum of
peak at RT= 5.237
1.20
1.00
Y
AU
0.80
3.00
0.60
0.40
2.00
AU
0.20
279.8
0.00
220.00
260.00
300.00
1.00
Z
340.00
380.00
nm
6.50
400.00
nm
0.15
0.10
Minutes
X
0.05
Uracile - 6.682
5.50
Myoglobin - 5.237
4.50
AU
3.50
IgG - 3.799
0.20
340.00
2.50
0.25
BSA - 4.280
280.00
Thyroglobulin - 3.079
XY->Chromatogram at 280 nm
220.00
0.00
2.50
3.50
4.50
5.50
Minutes
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6.50
7.50
Protein Analysis by UHPLC Lab Manual 2013,
Advanced Lab on Analysis on Bio-Molecules, Weizmann Institute, 2013
Peak identification by PDA is done by comparing a spectrum from an unknown peak to a spectrum from
a peak from a know standard who was stored in a user created library. A special algorithm in the
software defines the closeness of the spectra.
Fig 5:
The protein known markers‘ UV spectra are extracted and stored in a user created library.
The unknown peak’s spectrum is compared to these spectra and the closest one confirms the identity of
the unknown peak.
Fig 6:
Unknown’s UV spectrum is compared to the library to confirm its identity.
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Protein Analysis by UHPLC Lab Manual 2013,
Advanced Lab on Analysis on Bio-Molecules, Weizmann Institute, 2013
U-HPLC Instrument
Fig 7: The instrument is consisted of the following: Bottles of mobile phase, a pump, an autosampler, a
column manager/oven and a detector.
Fig.8:
Efficiency of the separation in UHPLC is higher than regular HPLC.
Analytical Benefits of U-HPLC





Higher Sensitivity
Separation between closely related compounds
Higher peak capacity
Shorter run times
Solvents and cost saving
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Protein Analysis by UHPLC Lab Manual 2013,
Advanced Lab on Analysis on Bio-Molecules, Weizmann Institute, 2013
References:
 "A REVIEW SIZE-EXCLUSION CHROMATOGRAPHY FOR THE ANALYSIS OF
PROTEIN BIOTHERAPEUTICS AND THEIR AGGREGATES", Journal of Liquid
Chromatography & Related Technologies, , 35:2923–2950, 2012, Paula Hong, Stephan Koza
& Edouard S. P. Bouvier,
(http://www.tandfonline.com/doi/pdf/10.1080/10826076.2012.743724) or
http://www.waters.com/webassets/cms/library/docs/720004595en.pdf

Introductory article in Hebrew: “http://www.forumsci.co.il/HPLC/ShulaUPLC.pdf

http://www.sepscience.com/Techniques/LC/Articles/172-/Size-Exclusion-Ultra-PerformanceLiquid-Chromatography-for-the-Analysis-of-Covalent-High-Molecular-Weight-Insulin
“Critical evaluation of fast size exclusion chromatographic separations of protein aggregates,
applying subof Pharmaceutical and Biomedical Analysis 78– 79 (2013) 141– 149


Waters’ Application Note:
http://www.waters.com/webassets/cms/library/docs/720004412en.pdf

“New trends in reversed-phase liquid chromatographic separations of therapeutic peptides and
proteins: Theory and applications", Szabolcs Fekete, Jean-Luc Veuthey, Davy Guillarme,
Journal of Pharmaceutical and Biomedical Analysis, October 2012, Volume 69, Pages 9–27
(http://dx.doi.org/10.1016/j.jpba.2012.03.024)

Presentation:http://www.waters.com/webassets/cms/events/docs/advances_in_uplc_systems_c
hemistries_application_biosimilars.pdf
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Protein Analysis by UHPLC Lab Manual 2013,
Advanced Lab on Analysis on Bio-Molecules, Weizmann Institute, 2013
Experiment
Goals:
- Run an unknown sample and determine the Molecular Weight of its component/s by comparing
them to previously injected markers whose molecular weights are known from which a calibration
curve are created.
- Identification of sample components using the Spectral Matching of the unknown peaks’ UV
spectra to a previously created UV spectra library from known markers.
- Quantify the sample components by comparing their areas to those of the previously injected
standard solutions of known concentrations.
Experiment Procedure
The group of users will receive a vial with protein markers to be used for all the group. In addition, each
student will assigned to an unknown. The protein markers of molecular weights and concentrations will
be injected and processed to create a MW based calibration curve, then a quantitative calibration curve as
well as store them in a UV spectral library.
Each of the students will inject his/her unknown to get its MW determination, to quantify and to identify
it.
The final report should include the following:
1. Introduction on the analytical techniques
2. Explanation of the experiment
3. The report on the MW determination, the identification by UV spectrum and quantitation by the
calibration.
Chromatographic Conditions:
Instrument:
Column:
H-Class Bio UPLC with PDA detector, Equipped with 5 mm Titanium Cell.
ACQUITY UPLC BEH200 SEC, 4.6 x 150 mm, 1.7 μm
Sample:
Mobile Phase:
Weak Needle Wash:
Strong Needle Wash:
Seal Wash:
Injection Volume:
Flow Rate:
Column Temp.:
Detection:
BEH200 SEC Protein Standard (P/N 186006518)
100 mM sodium phosphate, pH 6.8
100% UHPLC grade water
100% UHPLC grade water
90/10 water/methanol
2 μL
0.3 mL/min
30 ˚C
280 nm
Note:
-Recommended Flow Rate Range for maximum column lifetime: 0.1–0.4 mL/min
-Recommended Maximum Operating Column Backpressure: BEH200—7,000 psi for both 150 and 300
mm
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Protein Analysis by UHPLC Lab Manual 2013,
Advanced Lab on Analysis on Bio-Molecules, Weizmann Institute, 2013
1. Preparation of the Mobile Phase: 100mM Sodium Phosphate Buffer (in 500 mL)
- Weigh out 500 ± 0.02 grams of H2O into a 500 mL beaker.
- Weigh out 3.55 ± 0.02 grams of Sodium Phosphate dibasic and add to the 500 mL beaker.
- Weigh out 3.45 ± 0.02 grams of Sodium Phosphate monobasic and add to the 500 mL beaker.
- Stir for a minimum of 30 minutes then filter the solution through a 0.2 μm Millipore GV filter.
- Measure the pH and record the value for reference purposes (approximate pH: 6.8).
- Filter solutions through a compatible 0.2 μm or smaller pore size filter. The use of a sterile filtration
apparatus is recommended for buffers capable of supporting microbial growth.
Notes:
-
-
Solutions that are susceptible to microbial growth should be replaced at regular intervals to avoid
column contamination. Do NOT refill partially full SEC Eluent bottles with new Eluent. Rather,
when required use new bottle containing freshly prepared SEC Eluent.
Select solvent inlet filters that are compatible with solutions used, and clean or replace filters
regularly when using solutions that are susceptible to microbial growth.
2. Preparing the Proteins Markers (Ready mix for calibration curve):
Analyte
pI
MW
1. Thyroglobulin, 3 mg/mL
4.6
669,000
2. IgG, 2 mg/mL
6.7
150,000
3. BSA, 5 mg/mL
4.6
66,400
6.8, 7.2
17,000
N/A
112
4. Myoglobin, 2 mg/mL
5. Uracil, 0.1 mg/mL
BEH200 SEC Protein Sample Preparation:
Dissolve the BEH200 SEC Protein sample in 500 μL of 100 mM sodium phosphate buffer pH 6.8. Once
solubilized, do not freeze sample, store at 2–8 °C for no more than a week.
Protein mixture separation on an ACQUITY UPLC BEH200 SEC, 1.7 μm column: A typical
Chromatogram
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Operation of H-Class Bio by Empower software
1- Preparing the System for work (In case the system is not READY for WORK)
Login to Empower Software (User: system Password: manager) -> Run Samples
Select the project: SEC_BEH200_Weizmann_Lab and the system: H_Class_PDA
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The “Run Samples” screen is opened:
Click on Console icon
and the following window is opened: UPLC Console
Each system component is controlled by the “Control” menu.
Entire system:
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Pump (Solvent Manager):
Injector (Sample manager):
Detector:
USING THE SYSTEM STARTUP Tool (If required)
Transfer the A and/or B inlets into the Mobile phase Bottle.
 Go to the Console->Select to the ACQUITY UPLC SYSTEM->Control->Startup system.
 Fill the values in the screens of the SYSTEM STARTUP.
The System Startup primes in parallel the following:
 A; B; C; D;
 Purge solvent and Wash solvent;
 Seal Wash.
 Prepares the system to the next HPLC method (Equilibrate)
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2- RUNNING SAMPLES IN THE RUN SAMPLES WINDOW:
In the Run Sample window make sure to be in the “Samples” screen and click on the first icon at the top
to load the “Sample Set Method”.
Select: “Load using a previously created sample set method”
Select the method of your group
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Make sure the Run Samples look as the following:
-
The list contains the standard (Protein Markers Mix) and the unknowns,
The column is in the right position and it was selected properly
The pressure is not too high (typical pressure is shown in the graph)
The instrumental parameters are as required (look at the instrument method’s screens as shown in
the following graphs)
Checking the Instrument Methods parameters: Click on EDIT below the box of the Instrument
Method. Select Instrument Method: “SEC_Proteins_BEH200_PDA”
PUMP parameters: Acquity Quaternary Solvent Manager (QSM)
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Acquity Sample Manager (Autosampler); Flow-Trough Needle (FTN)
ACQ-CM: Column manager:
Make sure the temperature is according to the method and that the right position of the column is
specified (Check carefully!):
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PDA Detector:
The acquisition by Photo Diode Array detector will be done in two modes: one as 3 dimensional data
(including UV spectral information), marked as 3D data, and the other is as two dimensional data (Just a
basic chromatogram), marked as 2D channel. Check both screens that the right parameters are used.
3D Data:
2D Channels:
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Check that the Method Set “Sec_Proteins_BEH200_MW_PDA” in your Sample-Set Method contains
all the modules for Acquisition of data, Processing and Reporting:
In the Run Samples with your Sample Set Method -
Select-> “Edit Amounts” icon -> Make sure the right values of Molecular Weights at decreasing order
(why?) and Concentrations are filled in the Markers Mix’ row. Click OK to close the window.
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Click on the Flow link in the “Quaternary Solvent Manager” square to set the flow rate and select the
water (C) or the buffer reservoir (A), depending on the instructions you get in the lab:
Always start with flow rate of 0.1 mL/min then increase the flow to the working value (0.3 mL/min in
this case)
In the Run Samples Instrument Method’s Window select the Instrument Method
“SEC_Proteins_BEH200_PDA” . Click on SETUP to activate it.
Next click on “Monitor” to observe the baseline in the detector. The system is ready when you see a full
scale of x-10e-5 AU:
Make sure that baseline is stable, scale on ~10-5 AU in the PDA detector; pressures and temperatures are
stable.
Select “Run and Process” in the window and click on the Green button to start running the Sample Set
Method and get a group of chromatogram in the “Sample Sets” window of the project. The lines that
were injected are marked in red.
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Processing and Reporting the Data
In the lab we will go to “Browse Project”->Sample Sets-> Select your sample set-> select “Review”,
open your method set “Sec_Proteins_BEH200_PDA” if not already opened.
The 2D chromatogram should show the proteins’ peaks with their molecular weights marked on them.
Check the following:
 The right Method Set/Processing Method are opened “SEC_BEH200_MW“ for the 2D channel and
“SEC_BEH200_PDA“ for the extracted channel from PDA.
 Check the Processing Method Integration and other processing parameters:
o Integration events should be set according to the acquired channel
o Components Table contains all the components and Amounts are set properly.
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The Review of PDA Chromatogram with its UV spectra:
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The Review of SEC work chromatogram:

When all the Peaks are integrated properly save the (revised) Processing Method and exit the
Review window.
If a re-Processing is needed: Go back to the Sample Sets View->Select your Sample Set-> Tools>Process Select “Use Acquisition Method Set”, Click OK.
REPORTING RESUTLS
SEC Results
Go to the Results View-> Select your SEC Results
Tools -> Preview/Publisher-> Select the Report Method “SEC Proteins Weizmann Lab”
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to get the MW of the protein, and save your report as PDF file.
Report of the SEC run:
Save the report as PDF in the LAB’s directory: SEC_BEH200_Weizmann_Lab.
Results of the PDA Run
Select the results of the PDA identification and quantification
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Go to the Results View-> Select your Result-> Preview/Publisher-> Select the Report Method “PDA
Report”
to get the quantities and identification by library match of your proteins.
Report:
Save this report as PDF file as well.
As can be seen above: The reports show the results of molecular weights of the unknown samples, their
quantities by the calibration standards, and their identification by comparing them to a spectral library.
Use the two Reports from the experiment as part of your lab report.
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