Technical NotePharmaFluidicsThe micro-Chip Chromatography Company

Peptide sample enrichment using µPAC™ trapping columns

KEY WORDS

Micro pillar array column, µPAC™, Sample enrichment, Sample clean-up, Desalting, Trap and Elute, Trapping columns, Microfabrication,

Bottom-up proteomics, Routine analysis, Peptide separation, Nano LC, Capillary LC, RPLC-MS/MS

Introduction

The online preconcentration of analytes onto low volume trapping columns is a commonly used injection strategy in nano- and microbore LC-MS/MS analysis of complex peptide mixtures. Compared to direct injection onto the analytical column, a sample trapping approach provides several advantages. By e�ectively desalting and preconcentrating the analytes of interest onto the trap column, analytical column lifetime and work�ow throughput can be improved. The trapping con�guration allows e�ective removal of sample matrix components such as salts and contaminants which can interfere with downstream MS analysis, hereby increasing analytical column lifetime and at the same time improving detection sensitivity and the spectral quality that is generated for a sample. It also allows loading higher volume and dilute samples at a much higher �ow rate than what is feasible when working with a direct injection approach, having a positive e�ect on the LC-MS/MS duty cycle.

However, apart from providing sample clean-up and preconcentration, it is also of paramount importance to maintain chromatographic performance when combining an analytical column with a trapping column. A poor combination will result in reduced chromatographic performance, which will in turn a�ect the quality of the data that is generated. Trapping column dimensions and surface chemistry have to be selected carefully to match the analytical column that is used. Typically, trapping columns with a capacity factor slightly lower than the analytical column will result in the best chromatographic performance as analytes will experience a second preconcentration or refocusing

event when eluted onto the analytical column. This eliminates the detrimental e�ect of pre-analytical column connections or void volumes on the separation e�ciency.

Figure 1: Graphical illustration of a µPAC™ trapping column both on the out- (on

top) and inside (bottom), including the capillaries equipped with 1/16” �ttings.

Table 1: µPAC™ product speci�cations and characteristics.

μPAC™ Trapping column speci�cations

Channel widthChannel lengthChannel depth

Inter pillar distance

Bonded phasePillar bed volumeTotal volume

Connection tubing

2 mm

10 mm 20 μm

2.5 μmPillar diameter 5 μm

Surface morphology Super�cially porous - 0.3 μm porous layerPore size 0.010 - 0.020 μm / 100 - 200 A

C18 + endcapped 0.25 μl 1 μl

Fitting type 1/16" stainless steel male nut and ferrule (Valco compatible) 40 μm ID fused silica - 1/16" PEEK protection sleeve

Technical NotePeptide sample enrichment using µPAC™ trapping columns 02PharmaFluidics

The micro-Chip Chromatography Company

Compared to conventional packed bed nano LC columns, the stationary phase support of the µPAC™ columns consists of super�cially porous silicon pillars that have been modi�ed with a hydrophobic ligand (Octadecyl or C18), and therefore they have a slightly lower capacity factor. Combining traditional ‘fully porous’ packed bed trapping columns with a µPAC™ analytical column will consequently result in decreased chromatographic performance.

To support the use of the 50 and 200 cm long µPAC™ columns, a micromachined trapping column was developed with matching stationary phase support morphology. Optimal trapping con�guration and sample loading conditions have been evaluated and compared towards a direct injection strategy in terms of chromatographic performance and protein identi�cations that can be achieved in a typical bottom-up proteomics experiment.

Experimental

µPAC™ trapping column speci�cations

The PharmaFluidics µPAC™ trapping column has been carefully designed to match the sample capacity of µPAC™ analytical nano LC columns. Product speci�cations can be found in Table 1. The trapping column consists of a 1 cm long and 2 mm wide micro�uidic channel that has been etched out of a silicon wafer. This channel is �lled with 5 µm super�cially porous pillars, positioned in an equilateral triangular grid at an interpillar distance of 2.5 µm. The actual separation chip or column is provided in an Aluminum protective casing and has been equipped with 1/16” stainless steel HPLC �ttings to �t directly into standard HPLC switching valve ports (Figure 1). The column has a perfectly symmetrical design and can be used in a bidirectional trapping con�guration without any risk of harming the separation bed. Besides this “in-valve” trapping con�guration, internal reducing connection T-pieces can be ordered to facilitate a “vented-T” or inline trapping con�guration as well.

Experimental setup

Figure 2 gives a schematic overview of the trapping con�guration that was used. The µPAC™ trapping column was positioned in a 6-port switching valve in the column compartment of a Thermo Scienti�c™ Ultimate 3000 nano RSLC system and maintained at 30°C during the entire experiment. The nano LC system was con�gured to perform trap and elute injection of 10 µl of sample onto the trapping column. For the sample uptake, 10 µl of sample was drawn into a 20 µl sample loop before switching the injection valve and trapping the sample onto the trapping column with a loading solvent consisting of 99% water, 1% acetonitrile and 0.1 % tri�uoroacetic acid (TFA).

Figure 2: Schematic representation of the 6-port switching valve trapping

con�guration. During sample trapping or sample loading, the switching valve

is in position A. In this position, the sample is pumped at high �ow rate (10-20

µl/min) over the µPAC™ trapping column. Hydrophobic analytes are trapped or

concentrated onto the trapping column, salts and contaminants are washed

away. By switching the valve to position B, the trapping column is placed in

line with the analytical column. The analytical pump forms a mobile phase

solvent gradient with increasing elution strength that elutes the analytes in

the opposite direction towards the analytical column. This is done at �owrates

ranging from 0.1 to 2.0 µl/min.

Valve Position A - Sample Loading

Valve Position B - Gradient Elution

Analytical Pump

Analytical Pump

Analytical Column

Analytical Column

Waste

Waste

Loading PumpSample loop

Loading PumpSample loop

Trap

ping

Colu

mn

Trap

ping

Colu

mn

03PharmaFluidicsThe micro-Chip Chromatography Company

Technical NotePeptide sample enrichment using µPAC™ trapping columns

Loading pump �ow rates of 10 and 20 µl/min were evaluated and this at trapping durations ranging from 1 to 6 minutes which equals a total volume running through the column of respectively 10 to 60 µl. After trapping, the 6-port switching valve was switched to put the trapping column in line with the µPAC™ analytical column and reverse elution was performed using a 60 min non-linear solvent gradient at 1 µl/min. The nano LC system was coupled to a Thermo Scienti�c™ Orbitrap Elite™ hybrid Ion Trap-Orbitrap mass spectrometer by using a nanoFlex ion source. The analytical column (50 cm µPAC™ column) was connected to a New Objective PicoTip™ emitter and a grounded connection was provided between the outlet union and the mass spectrometer. The voltage required for electro spray ionization (ESI) was applied on a 50 µm through-bore stainless steel union through a liquid junction. LC-MS parameters can be found in Table 2.

Sample preparation

Thermo Scienti�c™ Pierce™ HeLa Protein Digest Standard (P/N 88328) was obtained from Thermo Scienti�c™ in lyophilized form. 20 µg of lyophilized peptide material was reconstituted in 10 µl of 0.1% TFA in LC-MS grade water to a concentration of 1, 5, 10, 50, 100, 200, 500, 1000 and 2000 ng/µl. Samples were spiked with Thermo Scienti�c™ Pierce™ Retention time calibration mixture (0.5 pmol/µl; P/N 88320) to a �nal concentration of 50 fmol/µl. Freshly prepared protein digest standard was used for each type of experiment.

Table 2: Overview of the LC-MS/MS settings.

Experimental

MS settings

MS1

MS2

LC

Source

LC systemColumnMobile phase

Loading bu�er

Gradient pro�lesTemperatureSample

Injection

ESI source

ESI emitterPolarityCapillary temperatureSpray voltage positive ion

MS instrumentAcquisition mode

Resolution

Maximum ITScan range

AGC target

Maximum ITTopNIsolation windowNCECharge states rejectedCharge states not rejectedDynamic exclusion

Lock mass

Thermo Scienti�c™ Ultimate 3000 nano RSLC

PharmaFluidics 50 cm µPAC™ C18 A; Water (100%) with 0.1% (v/v) FA B; Water/acetonitrile (20/80) with 0.1% (v/v) FA Water/acetonitrile (99/1) with 0,1% (v/v) TFA

Loading pump �ow rate 10 - 20 µl/minAnalytical pump �ow rate 1.0 µl/min

non-linear 1-45% B in 60 min gradient 30°C 1-5-10-50-100-200-500-1000-2000 ng/µl HeLa cell digest

1 µl full Loop injection (direct injection) or 10 μL user de�ned (trapping)

Thermo Scienti�c™ nanospray ion source

New Objective PicoTip™ Emitter SlicaTip™ (FS360-20-10-CE-20-C10.5) Positive 275°C 2500 V

Thermo Scienti�c™ Orbitrap Elite™ DDA

60,000

500 msAGC target 1,00E+06

375 - 1500 m/z

unasigned and 12,3,4+20 sec

445.120028 Da (polysiloxane peak) (internal calibration)

250 ms162.0 m/z

1.00E+05

35

with 50 fmol/µl Thermo Scienti�c™ PRTC Mixture

Figure 3: Schematic representation of a diluted sample that will be

concentrated on a µPAC™ trapping column during a 6 min loading window.

A 20 µl plug originating from the sample loop is indicated in dark blue, while

the analytes are shown in orange (and yellow for the most hydrophilic ones)

in a 1µl volume of the entire trapping column (including capillaries).

The �ow through volume that passed through the column over time is

indicated on the right side.

Wash Volume - To Waste

Sample Loop - 20 µl

Transfer Tubing - 3.5 µl

µPAC Trapping Column - 1 µl

2 Min Trapping - 20 µl

1 Min Trapping - 10 µl

Sample Uptake

3 Min Trapping - 30 µl

4 Min Trapping - 40 µl

5 Min Trapping - 50 µl

6 Min Trapping - 60 µl

04PharmaFluidicsThe micro-Chip Chromatography Company

Technical NotePeptide sample enrichment using µPAC™ trapping columns

Results

The main goal of this technical note is to illustrate the use of a µPAC™ trapping column in combination with a µPAC™ analytical column. The focus lies on the experimental setup and preconcentration of diluted samples, rather than on desalting and sample clean-up. A head-on comparison between direct injection and trapping was made as well as an evaluation of di�erent trapping times.

Peptide Recovery

To evaluate the relative peptide recovery that can be achieved for di�erent sample loads and at di�erent trapping times, a de�ned concentration range of HeLa cell lysate digest samples was prepared (10-50-100-200 ng/µl) and injected onto the trapping column, trapped for a de�ned duration (1-2-3-4-5 and 6 min) at 10 µl/min and eventually eluted towards the analytical column and analyzed by MS/MS. The volumes of the trapping setup have been schematically represented in Figure 3. The position of the sample and the total volume of sample loading solvent that has �own over the trapping column towards waste have been

illustrated. Three di�erent peptides originating from the HeLa cell lysate digest were monitored and relative peak areas have been compared to determine the optimal trapping conditions for maximal peptide recovery. Extracted ion chromatograms obtained for these 3 peptides (Figure 4) clearly show that these peptides have been selected over a range of hydrophobicities (spread out over the elution window) to get a good representation towards complex protein digest samples. For sample concentrations up to 100 ng/µl, corresponding to a sample load of 1 µg of sample on-column, optimal peptide recovery was obtained between 2 and 4 min trapping at 10 µl/min. 3 to 4 min of trapping are considered more favorable to make sure that the total loop volume of 20 µl and some additional wash volume has been pumped over the µPAC™ trapping column. At trapping times beyond 4 min, peptide peak areas start to decrease, especially for the most hydrophilic peptide. The loading capacity of the µPAC™ trapping column is determined between 1 and 2 µg of protein digest sample, as the most hydrophilic peptide that was monitored is clearly lost when 2 µg of protein digest is trapped beyond 3 min. In order to have a good balance between sample recovery and sample clean-up, 4 min trapping at 10 µl/min and 2 min at 20 µl/min have been used for all further trapping experiments.

Figure 4: Extracted ion chromatograms of three di�erent peptides originating from the HeLa protein digest sample (yellow: low hydrophobic peptide; orange: medium

hydrophobic peptide and red: highly hydrophobic peptide) are shown on top. At the bottom the normalized peak areas of each of these peptides are plotted for

di�erent trapping times (1 to 6 min) and sample concentrations (100-2000 ng HeLa digest).

0

20

40

60

80

100

120

1 2 3 4 5 6

100 ng HeLa digest

0

20

40

60

80

100

120

Norm

aliz

ed P

eptid

e Pe

ak A

rea

[%]

0,0E+00

1,0E+07

2,0E+07

3,0E+07

4,0E+07

5,0E+07

6,0E+07

14 15 16 17 18 19 20 21 22 23

Retention Time [min]

Rela

tive

Abun

danc

e [/]

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aliz

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eptid

e Pe

ak A

rea

[%]

1 2 3 4 5 6

500 ng HeLa digest

0

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1 2

Norm

aliz

ed P

eptid

e Pe

ak A

rea

[%]

23 240,0E+00

1,0E+07

2,0E+07

3,0E+07

4,0E+07

25 26 27 28 29 30 31 32Retention Time [min]

Rela

tive

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e [/]

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1000 ng HeLa digest

0

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1 2 3

Norm

aliz

ed P

eptid

e Pe

ak A

rea

[%]

2000 ng HeLa

34 350E+00

5E+06

1E+07

2E+07

37 38 39 40 41 42 43Retention Time [min]

Rela

tive

Abun

danc

e [/]

4 5 6

ng HeLa digest

4544 46 4733

Trapping Duration [min] Trapping Duration [min] Trapping Duration [min] Trapping Duration [min]

05PharmaFluidicsThe micro-Chip Chromatography Company

Technical NotePeptide sample enrichment using µPAC™ trapping columns

Figure 6: Extracted ion chromatograms for a 10 µl direct injection mode (on

top) and a 10 µl “in-valve” trapping con�guration both separated according

the same 60 min solvent gradient, indicating the increased throughput as well

as the sample volume �exibility when using a µPAC™ trapping column.

Figure 5: Average peak widths at FWHM for each of the 15 PRTC peptides that

have been spiked at 50 fmol/μl into a HeLa cell lysate digest. Green bars

represent the setup without trapping (direct injection mode) while blue bars

represent the setup with an “in-valve” µPAC™ trapping column. Mean values

from all selected peptides were shown on the right side of the graph.

Chromatographic Performance

Besides good peptide recovery over a range of trapping times, excellent peak shapes were found when using the µPAC™ trapping column with an “in-valve” trapping setup. Based on 15 reference peptides from the Pierce™ Retention time calibration mixture, average peak widths of 0.25 min (FWHM) were observed over the entire elution window (Figure 5).

Compared to the direct injection experiments, the use of a µPAC™ trapping column resulted in a 5% increase in average peak width. Plotting the mean peak widths (n=3) for each of the 15 reference peptides clearly shows that there is no bias of peak broadening towards hydrophilic nor hydrophobic peptides.

Increased Throughput for high volume injections

The positive e�ect of the trapping con�guration on the LC-MS/MS method throughput is illustrated in �gure 6, where basepeak chromatograms obtained for the separation of 500 ng of HeLa digest have been compared. In the upper chromatogram, 10 µl of sample (50 ng/µl HeLa digest) has been injected onto the column using a 20 µl sample loop in direct injection mode. The bottom case shows the same injection, but then performed with a 4 min “in-valve” trapping method at 10 µl/min. As these separations were all accomplished at an analytical �ow rate of 1 µl/min, the e�ect of injecting a 20 µl sample loop in direct mode will be even more pronounced when operating the analytical column at typical nano LC �ow rates in the range of 300 nl/min. 20 µl direct injection was only used to illustrate the e�ect on sample loading time. For all other direct injection experiments, a 1 µl sample loop with concentrated (10x) samples was used.

Proteome coverage

Acquired MS2 spectra were screened against the human reference database (UniProt) with a false discovery rate (FDR) of 0.1% by using the Thermo Scienti�c™ Proteome Discoverer 2.2 platform. The number of identi�cations from a direct injection (1 µl full loop) of a concentrated sample were compared against a tenfold diluted sample (10 µl sample in 20 µl sample loop) by using the “in-valve” trapping setup (Figure 7). Using the direct injection method as reference, on average 88% of the Peptide-Spectrum Matches (PSM’s), 84% of the peptide and 90% of the protein identi�cations could be observed by performing “in-valve” trapping of a tenfold diluted sample. On the low side of the concentration range, using a µPAC™ trapping column allows identifying over 2340 peptides and 780 protein groups for a protein digest sample with a concentration of barely 1 ng/µl. Up to a concentration of 100 ng/µl (or 1 µg of sample injected) the number of peptide and protein identi�cations’s increases with the amount of sample that was loaded, yielding consistent identi�cation of 5120 peptide and 1475 protein

0,10

0,15

0,20

0,25

0,30

0,35

0,40

1 3 5 7 9 11 13 15 MEAN

PRTC peptide

direct injection

with µPAC™ trap

Aver

age

Peak

Wid

th FW

HM [m

in]

0,0E+00

2,0E+07

4,0E+07

6,0E+07

8,0E+07

1,0E+08

0 10 20 30 40 50 60 70 80 90

Retention Time [min]

Rela

tive

Abun

danc

e [/]

0,0E+00

2,0E+07

4,0E+07

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8,0E+07

1,0E+08

0 10 20 30 40 50

Retention Time [min]60 70 80 90

Rela

tive

Abun

danc

e [/]

06PharmaFluidicsThe micro-Chip Chromatography Company

Technical NotePeptide sample enrichment using µPAC™ trapping columns

groups. Loading more than 2 µg of protein digest sample did not result in a signi�cant increase in identi�cations, which may well be a consequence of the fact that early eluting peptides tend to get lost when trapping 2 µg of protein digest sample.

E�ect of trapping �ow rate

When elevated �ow rates (20 µl/min) were used to load the sample onto the µPAC™ trapping column (trapping volumes were kept constant), 5% less protein groups and 10% less peptide groups were identi�ed (Figure 8). Despite the small loss in absolute identi�cations when operating the µPAC™ trapping column at elevated �ow rates, loading times can be signi�cantly reduced when analyzing larger sample volumes. As shown in Figure 9, running the trapping column continuously for over a month did not result in signi�cant column backpressure changes. Operating the trapping column at 10 µl/min, a backpressure of 18 bar is observed, and for 20 µl/min this is 36 bar. Even though the maximum operating pressure is de�ned at 350 bar, no serious improvements in throughput nor in chromatographic performance and experimental outcome are expected by increasing the loading pump �ow rate beyond 20 µl/min.

Figure 7: Average numbers of identi�ed protein and peptide groups as well as

the number of PSM’s for di�erent sample concentrations. The left bars (green)

represent the direct injection of 1 µl (sample concentrations 10, 50, 100, 500,

1000 ng/µl), while the right bars (blue) characterize the �ndings obtained

with an “in-valve” µPAC™ trapping setup where tenfold diluted samples were

injected (1, 5, 10, 50, 200 ng/µl).

977

1,26

3

1,36

1 1,55

5

1,60

7

782

1,17

4

1,22

7 1,44

8

1,47

5

1,46

7

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1000

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Num

ber o

f Pro

tein

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ups

10ng 50ng 100ng 500ng 1000ng 2000ng

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4,74

3 5,43

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5,80

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2,34

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4,03

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5,12

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4,88

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0

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Num

ber o

f Pep

tide

Grou

ps

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3,32

1

5,28

8 6,19

3 6,81

1

6,87

9

2,68

5

4,50

8

5,82

9

6,02

2

6,24

5 6,82

2

0

1000

2000

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4000

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6000

7000

8000

Num

ber o

f PSM

10ng 50ng 100ng 500ng 1000ng 2000ng

Figure 8: Bar chart representing the relative number of peptide (top) and

protein (bottom) groups that could be identi�ed when operating the µPAC™

trapping column at 10 µl/min (green) or at 20 µl/min (blue) for di�erent

sample concentrations ranging from 100 ng to 3000 ng Hela cell lysate digest.

100%

100%

100%

100%

100%

89% 95

% 98%

98%

96%

0%

25%

50%

75%

100%

125%

150%

Rela

tive

num

ber o

f pro

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gro

ups

100ng 500ng 1000ng 2000ng 3000ng

10µL/min 20µL/min

100%

100%

100%

100%

100%

80%

94%

93%

92%

87%

0%

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ups

100ng 500ng 1000ng 2000ng 3000ng

10µL/min 20µL/min

07PharmaFluidicsThe micro-Chip Chromatography Company

Technical NotePeptide sample enrichment using µPAC™ trapping columns

Conclusions

µPAC™ trapping columns can be e�ectively used to perform large volume (≥5 µl) peptide sample injections on µPAC™ analytical columns with minimal impact on the total analysis time. The unique design of these trapping columns allows loading samples at high �ow rates (10-20 µl/min) and this at very low operating pressures (20-40 bar). The surface morphology of these columns is designed to match µPAC™ analytical nano LC columns (50 and 200 cm long nano LC versions available). Minimal loss of chromatographic performance was observed when comparing the “in-valve” trapping setup to a direct injection strategy. For 15 PRTC peptides, on average 5% increase in peak width (FWHM) was observed. This can be attributed to the unique properties of the µPAC™ trapping column, with a super�cially porous or core-shell stationary phase morphology that matches perfectly with the retentive properties of the µPAC™ analytical columns. Up to a column load of 1 µg

protein digest sample, optimal peptide recovery was observed when 30 to 40 µl of loading solvent was pumped towards the µPAC™ trapping column. When loading 2 µg of protein digest sample, the sample capacity of the µPAC™ trapping column is exceeded and early eluting peptides are washed away during trapping. This is also represented by the number of peptide and protein identi�cations obtained when performing “in-valve” trapping at 10 µl/min for 4 min. By performing trapping of tenfold diluted samples, on average 84% of the peptides and 90% of the protein groups that were identi�ed with a direct injection strategy could be identi�ed.

Figure 9: Overview of µPAC™ trapping column back pressure during the performed experiments when operating at 10 µl/min (green) and 20 µl/min (blue).

No signi�cant increases in back pressure were observed.

0 10 20 30 40 50 60

10uL/min

20uL/min

Trap

ping

Colu

mn

Pres

sure

[bar

]

Injection Number

0

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08PharmaFluidicsThe micro-Chip Chromatography Company

Technical NotePeptide sample enrichment using µPAC™ trapping columns

Key features of the µPAC™ Trapping column

Flow rate �exibility:0.1 to 20 µL/min – corresponding column backpressures of respectively 0 and 40 bar – maximum operating pressure is 350 bar.

Column robustness: Each column has been manufactured by etching channels out of a solid piece of silicon, the column is perfectly bidirectional and contains no particles nor frits.

Column to column reproducibility:Each column is manufactured using the same lithographic mask, making every column identical.

Sample preconcentration:Preconcentration of diluted samples or samples with very low peptide content into a sub-microliter trapping volume.

Decrease sample loading times:Depending on the analytical column �ow rate; 10-50 fold reduction of the sample loading time for high volume (5 – 20 µl) samples.

Sample clean-up:E�ective removal of salts, reagents and contaminants that have no a�nity for hydrophobic bonded phases. Optimal compatibility towards µPAC™ analytical columns:Minimal loss of chromatographic performance due to matching stationary phase morphologies.

µPAC™ driven separations – Better by Design Conventionally LC columns are fabricated by stacking (packed beds) or depositing (monoliths) material into a capillary. PharmaFluidics’ µPAC™ technology (micro Pillar Array Column) is unique in its kind as it is built upon the precise micromachining of designed chromatographic separation beds into silicon. This approach brings along three crucial and unique characteristics:

Perfect Order.µPAC™ beds are designed with a high degree of order, eliminating heterogeneous �ow paths otherwise present in conventional columns (so called Eddy dispersion). Flow through µPAC™ columns adds very little dispersion to the overall separation. As a result, peaks remain sharper and sensitivity is increased.

High Permeability.µPAC™s operate at moderate pressures, typically lower than 300 bar. Separation channels with exceptional length (50 cm to 200 cm) are therefore possible. These are folded onto a small footprint by a interconnecting concatenating bed segments.

Solid Backbone.The micromachined backbone of the separation bed forms a rigid structure that is not in�uenced by pressure. There are no obstructions by touching surfaces, and there is no risk for perturbations by pressure �uctuations.

Technologiepark-Zwijnaarde 82B-9052 Ghent (Zwijnaarde)Belgiumwww.pharmafluidics.cominfo@pharmafluidics.com

Katrien Vanhonacker, MSc, MBAVP Business Development & Sales

katrien.vanhonacker@pharmafluidics.comM: +32 476 85 38 76

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