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  • Published: 13 March 2018

Size Exclusion Chromatography Method for Purification of Nicotinamide Mononucleotide (NMN) from Bacterial Cells

  • George Cătălin Marinescu   ORCID: orcid.org/0000-0001-9429-4502 1 , 2 ,
  • Roua-Gabriela Popescu 1 , 2 &
  • Anca Dinischiotu 1  

Scientific Reports volume  8 , Article number:  4433 ( 2018 ) Cite this article

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  • Analytical chemistry
  • Small molecules

Over 12% of the world’s health resources are spent on treating diabetes, as high blood glucose is the third cause of mortality worldwide. Insulin resistance is the basis of the most common form of diabetes: type 2 diabetes. Recent animal studies report successful attempts at reversing type 2 diabetes by the administering of the NAD + precursor nicotinamide mononucleotide (NMN). However, the current high price of this molecule urges for more efficient and cost-effective production methods. This work proposes a method for purifying NMN by Size Exclusion Chromatography (SEC) on silica with a covalently attached coating of poly(2-hydroxyethyl aspartamide) (PolyHEA) stationary phase using an isocratic elution with a denaturing mobile phase (50 mM formic acid) from a complex molecular mixture such as a fermentation broth. The eluted peaks were identified by UV-Vis analysis and confirmed with ESI+ mass spectrometry and a HPLC reversed-phase method. The proposed SEC method is simple, patent-free, directly applicable for industrial production with a minimum scale up effort. The need for multiple chromatographic steps is eliminated and the lysate filtration and clarification steps are simplified. Substantial reduction in NMN production costs and increased purity of NMN to the level suitable for usage in humans are expected.

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Introduction.

High blood glucose level is the third highest risk factor for premature mortality worldwide. The more common condition, type 2 diabetes, has increased with modern life, urbanisation, reduced physical activity and an ageing population. It affects the blood vessels, the heart, kidneys and nerves, eventually resulting in disability and premature death 1 , 2 . Previously, an association between insulin resistance and type 2 diabetes has been recognised 3 . Moreover, abnormal mitochondrial function was correlated with type 2 diabetes 4 , 5 , 6 , 7 , 8 .

In this context, gene expression and proteomics studies revealed a correlation between insulin resistance and a down-regulation of protein complexes involved in mitochondrial oxidative phosphorylation 9 , 10 , 11 . Lower expression of mitochondrial complexes I-IV influences the redox state of the cell, diminishing ATP production and antioxidant capacity. In all the organs of 12 months old Wistar rats, an increase of p53 acetylation, a decreased activity of nicotinamide adenine dinucleotide (NAD + ) dependent sirtuin-1 (SIRT1) and a mild over-expression of SIRT1 was correlated with diminished NAD + level and NAD + /NADH ratio 12 . Although NAD + is recycled by the salvage pathways 13 , 14 , 15 , it is consumed by poly(ADP-ribose) polymerases (PARPs) and glycohydrolases (CD38 and CD157) 16 and therefore therapeutic interventions to restore NAD + level are crucial. Nicotinamide mononucleotide (NMN), a polar molecule with a molecular weight of 334 Da 17 , was proven effective as an NAD + precursor in glucose intolerance studies, restoring NAD + levels in mice with type 2 diabetes induced by a high fat diet or aging 18 . The nuclear NAD + level modulates mitochondrial encoded gene expression and mitochondrial homoeostasis through a new pathway regulated by SIRT1 19 . NMN was also recently found useful in limiting brain injury following intracerebral hemorrhage 20 .

In the past, NMN was prepared by incubation of diphosphopyridine nucleotide with potato pyrophosphatase 21 or from nicotinamide by extracts of acetone-powered human erythrocytes 22 . These methods produced low quantities of NMN. Nowadays NMN is obtained by microbial biotechnology techniques. To reduce the high cost of NMN and to improve on the available purity requires innovation and optimisation of the current production methods. In this study the focus was directed on the downstream part of the biotechnological process. Only one purification method for NMN, utilising of ion exchange chromatography (IEC) followed by precipitation in acetone 23 was identified in the scientific literature. General methods for nucleotides and other small molecules purification from biological extracts proposed separation in formic acid followed by filtration, lyophilisation, and two-dimension high performance liquid chromatography (HPLC) methods: boronate-affinity and ion-pair 24 , or reversed-phase chromatography (RPC) or via high speed counter-current chromatography (HSCCC). Several analytical HPLC methods have been reported using RPC for NMN quantification on either C18 coated silica 25 or porous graphitic carbon stationary phase 19 , 26 . A similar approach, but using a gradient elution on a RP XBP C18 column (100 mm × 2.1 mm, 5 μm) and positive Electrospray Ionisation (ESI+) mass spectrometry detector running in Selected Ion Monitoring mode was previously reported for quantification of a NMN related molecule, also a NAD + precursor: Nicotinamide Riboside (NR) 27 .

The goal of this study was to design a simple and cost effective NMN purification method from a complex molecular mixture, such as the lysate of E. coli strain BL21 (DE3) pLysS genotype E. coli B F–dcm ompT hsdS(rB– m B–) gal λ(DE3) [pLysS Cam r ] [pET28a-hdNadV Km r ].

Single step NMN chromatographic separation by SEC-HPLC

The SEC-HPLC protocol was performed as explained in the Methods section employing a clarified bacterial lysate sample and standard NMN (Fig.  1A,C and E ). Analysing the Photodiode Array (PDA) data led to the identification of the retention time (RT) for NMN as 6 minutes. A clear separation of NMN from other molecules of lysate is shown (Fig.  1B and D ). The most abundant contaminant in the lysate had a retention time between 3.5 and 4 min (Fig.  1F ) which corresponds to the impurity peak identified in the NMN standard at the same RT (Fig.  1E ).

figure 1

HPLC SEC separation of nicotinamide mononucleotide (NMN). The light absorption spectra (200–600 nm) ( A , B) , (200–300 nm) ( C , D ) were recorded by the Jasco Photodiode Array (PDA) detector. ( E , F ) represent 260 nm chromatograms extracted from above PDA data. Mobile phase was 50 mM formic acid, with a flow rate 3 mL/min, the stationary phase was PolyHEA, column dimensions: 250 × 9.4 mm; 5 μm, 60-Å. Sample volume was 20 μL of 5 mM nicotinamide mononucleotide standard (Sigma Aldrich, however impure, as shown) ( A , C , E ), respective bacterial cell lysate ( B , D , F ). NMN elutes at 5.7 minutes and is clearly separated from other similar molecules (e.g. nicotinamide which has a similar light absorption spectrum, is more abundant in the bacterial lysate and elutes at 3.8 minutes). The second polynomial order calibration curve with a correlation coefficient of 0.99 was obtained for quantitative determination by integration of the peak area of 5 NMN samples with known concentration levels: 0.03; 0.16; 0.33; 1.67; 3.34 g/L ( G ).

The chromatogram obtained at 260 nm was extracted from the PDA data and used for quantification by peak area integration (Fig.  1E ).

For accurate NMN quantification, the calibration method described in the Methods section was used. A second order polynomial equation was obtained with the coefficient of correlation of 0.99 (Fig.  1G ), covering samples with NMN concentrations from 0.03 to 3.34 g/L.

Mass spectrometry identification of NMN

Although UV-Vis light absorption data recorded by the HPLC PDA detector provide spectrum information for NMN identification, to eliminate any doubt related to the correct peak identification, a mass detector was used. The Total Ion Current (TIC) chromatogram (Fig.  2A ) resulting from running the protocol described in section Size Exclusion Chromatography High Performance Liquid Chromatography (SEC-HPLC) (NMN standard sample, SEC protocol on PolyHEA column) shows a peak at 6 minutes, thereby correlating with the HPLC data (Fig.  2B ). The MS spectrum of the NMN separated from the bacterial lysate (Fig.  2H ) is similar to the spectrum of the NMN standard (Fig.  2G ). The specific m/z values (shown on both standard and lysate separations) are: 335 for NMN (molecular single protonated ion [M + H] + ); 669 for double molecular NMN single protonated ion [2 M + H] + ; 123 for the [M + H] + nicotinamide (NAM) ion, resulting from ion-source fragmentation of NMN. Selecting only m/z values between 334.5 and 335.5 (Fig.  2E ) from the full MS data recorded while running the bacterial lysate separation by SEC on a PolyHEA column, a clear peak having the same RT as 260 nm chromatograms of the NMN standard (Fig.  2B ) and bacterial lysate sample (Fig.  2D ) is shown. The most abundant contaminant from the bacterial lysate was simultaneously identified on: TIC chromatogram (Fig.  2C ), 260 nm chromatogram (Fig.  2D ) and the MS chromatogram of the ions having an m/z between 122.5 and 123.5 (Fig.  2F ) at a retention time of 4 min. The molecular identity of collected NMN fractions was confirmed by comparison against standard NMN MS/MS spectra (Fig.  3 ).

figure 2

Mass spectrometry identification of nicotinamide mononucleotide (NMN). Thermo Velos Pro MS detector ESI + mode, Spray Voltage: 3 kV, Capillary temp: 375 °C; isocratic HPLC elution with 50 mM formic acid flow: 3 mL/min, PolyHEA column 250 × 9.4 mm, 5 μm, 60-Å. Sample volume: 20 μL. H-ESI-II ion source was connected parallel to the MD2015 UV-Vis detector using 1:10 post column flow splitter. Standard NMN 5 mM (Sigma Aldrich) sample elution: total Ion Current chromatogram ( A ) shows NMN eluting at 6 minutes. 260 nm Chromatogram ( B ). NMN is identified by protonated molecular ion [M + H] + (335 Da) on the MS spectrum at a retention time of 6.07 ( G ), double molecular protonated ions [2 M + H] + (669 Da) and nicotinamide (NAM) fragment protonated molecular ion (123 Da) resulted from NMN fragmentation inside the ionisation source. Bacterial lysate: Total Ion Current ( C ); HPLC UV detector 260 nm, analogue input ( D ); m/z 335 mass chromatogram showing NMN peak at RT: 5.61 ( E ); m/z 123 mass chromatogram showing NAM peak at RT: 3.65 ( F ); NMN MS spectrum showing protonated molecular ion [M + H] + (335 Da), double protonated molecular ion [2 M + H] + (669 Da) and the nicotinamide protonated molecular ion (123 Da) known to result from NMN fragmentation inside the ionisation source ( H ).

figure 3

MS/MS Collision-Induced Dissociation (CID) spectral data of nicotinamide mononucleotide (NMN) from collected fractions (SEC and RPC) against standard NMN, using Thermo Velos Pro linear ion trap MS detector running in ESI+ mode, Spray Voltage: 3 kV, Capillary temperature: 375 °C, flow: 3 µL/min, H-ESI-II ion source.

Purity evaluation of NMN separated by SEC-HPLC using reversed-phase HPLC

The NMN RT using the RP-HPLC protocol was 3 minutes and the purity was determined as a percentage of the NMN peak area relative to a total detected peaks area of 260 nm chromatogram (Table  1 ). On the recorded 3D PDA absorption spectrum (Fig.  4A ) there was no detectable signal above 280 nm therefore a plane view of the interval between 200 and 300 nm was generated. This result highlights a contaminant eluting at RT 4 min (Fig.  4C ) which is clearly represented on the 260 nm chromatogram (Fig.  4E ). The 3D PDA absorption spectrum of the NMN obtained from the bacterial lysate by the proposed SEC method (as shown in section Mass spectrometry (MS) ) has no detectable impurity (Fig.  4B,D and F ).

figure 4

Purity evaluation of nicotinamide mononucleotide (NMN) separated by SEC PolyHEA using reversed-phase HPLC (C18). The light absorption spectrum recorded by the Jasco MD2015 UV-Vis Photodiode Array (PDA) detector during HPLC isocratic elution, NMN standard Sigma (however, impure as shown) ( A , C ) versus NMN purified from bacterial lysate by SEC PolyHEA ( B , D ). HPLC Chromatogram 260 nm isocratic elution of NMN standard Sigma 95% purity ( E ) versus NMN purified from the bacterial lysate by SEC PolyHEA ( F ). Mobile phase: 10% acetonitrile, flow rate 0.9 mL/min, column: Genesis C18 James Column 250 × 4.6 mm, 4 µm, 120 Å, injected sample: 20 μL. 10 mM NMN, starts eluting at 3 minutes.

Endotoxin content in NMN fractions

The endotoxin level in the NMN fractions purified by SEC method was 0.51 EU/mL. This is below the calculated acceptable parenteral administration limit (1.18 EU/mL) (Fig.  5 ).

figure 5

Endotoxin detection in NMN fractions by the LAL chromogenic method. The concentration of NMN sample was 1 mM.

Our attempt to reproduce the previously reported ion exchange method 23 of NMN separation from the bacterial lysate did not produce the expected results, probably because of the high nicotinamide (NAM) content of our bacterial lysate, as growing media was supplemented with 1% NAM as substrate for Nicotinamide phosphoribosyl transferase (Nampt). The mass chromatogram of ions with an m/z between 122.5 and 123.5 (Fig.  2F ) corresponding to the known m/z value for NAM [M + H] + ion correlates with the RT of the most abundant impurity from the lysate (Fig.  2D ). NAM and NMN co-eluted in the Dowex Cl − Ion Exchange Chromatography (IEC) column and co-precipitated in acetone together with high amounts of salts (data not shown). As previously reported, IEC optimisation is a time consuming and expensive process 28 , thus efforts were spent on identification of a simpler method.

Often used for analytical purposes, RP-chromatography (RPC) was not the most desirable method for separating hydrophilic molecules because they are not well retained by the stationary phase 29 . Previously, RPC used for NMN quantification by Triple Quadrupole LC/MS/MS 25 provided excellent analytical quantitative data. However, the results were obtained by RPC separation and selective data acquisition (multiple reaction monitoring) of the m/z values corresponding to the molecules of interest including NMN, but ignoring other possible co-eluting contaminants. This is adequate for analytical quantification but not suitable for preparative purposes. Selected-ion monitoring (SIM) filters out all information on the co-eluting molecules, and was therefore inadequate for preparative purposes. Although the further purification of the NMN standard by RPC was successful (Fig.  4C and E ), the small RT difference between the two peaks limited the preparative usage of the method, while higher column load widened the peaks and decreased resolution. Since the most abundant impurity in our bacterial lysate was NAM (Figs  1 F and 2D,F ) and it had a similar RT with the main impurity from the NMN standard (Fig.  1E ) on the proposed SEC method, it was thus concluded that the detected impurity in the NMN standard was also NAM. Therefore, the unknown peak (RT = 4 min) on the RP elution (Fig.  4C and E ) of the NMN standard could be NAM. Both NAM and NMN peaks resulted by RP separation were wider than the peaks resulting from the proposed SEC method, generating a lower NMN concentration in the collected fraction and a higher likelihood of co-eluted contaminants.

The most interesting and newest RPC methods providing good RT and resolution for NMN separation use porous graphitic carbon 19 , 26 . The main drawback of this technique is the high price tag of this material which makes its usage prohibitive for preparative applications. A previously reported method for separation of nucleotides from whole-cell extract by formic acid extraction, filtration followed by a two-step chromatographic process: boronate-affinity and ion-pair 24 , facilitated a good separation for NMN. This, however, had the disadvantage of the two-step chromatographic process, that are more expensive compared to our proposed single step SEC.

The key of the proposed SEC separation is the stationary phase: silica with a covalently attached coating of poly(2-hydroxyethyl aspartamide) (PolyHEA). When using a non-denaturing mobile phase, the hydrogen bonds between adjacent polymer chains of the coating make the coating impermeable. When a denaturing mobile phase (like 50 mM formic acid) is used, the chaotropic agent prefers the stationary phase instead of water to form hydrogen bonds. The increased steric radius of the PolyHEA coating might occlude the 60 Å pore, but by disrupting the hydration layer the available pore volume increases and the space between polymer chains becomes accessible to the mobile phase. This distance now represents the effective pore diameter 30 . When a non-denaturing mobile phase is used with a 60 Å pore PolyHEA column, the separation range is 60–10000 Da (according to manufacturer’s manual). Using the denaturing mobile phase, the separation range shifts to 20–600 Da, which makes it suitable for separation of NMN (334 Da) from other small solutes. This is the only SEC stationary phase (according to our knowledge) having a resolution in this range.

SEC was used for two reasons: to desalt the sample to use the electrospray ionisation mass spectrometer and to filter out the higher molecular weight compounds from the mixture. Our first intention was to collect the fraction containing NMN, to identify the co-eluting contaminants and further purify it by a second chromatographic method. It was a surprise to discover that this material made unnecessary any additional purification steps. Surprisingly, using the new method, further purification of the standard NMN (Sigma Aldrich, 95% advertised purity) was made possible as illustrated in Fig.  1E . Due to the known high NAM content of the bacterial lysate, the main contaminant in the lysate was identified as NAM, which has been determined to have a retention time of 3.5–4 minutes (Fig.  1F ).

The data generated revealed that the elution order between NAM (122 Da) and NMN (334 Da) was surprisingly reversed. According to the SEC principle, NMN should elute first, having a greater molecular weight, suggesting that some other physical interactions occur 31 . Electrostatic charge might be the cause of the observed reversed elution order, as the titration curve of the PolyHEA material showed that at a pH below 4.4 the material behaves as having a net positive charge, and above 4.4 it behaves as though having a negative charge 30 . With 50 mM formic acid as the mobile phase, the pH was 2.0, therefore the coating net positive charge might cause electrostatic repulsion of NAM (pKa = 3.63) 32 which also has a net positive charge at pH 2.0. The NMN phosphate group might have formed an electrostatic attraction with the coating. To verify this hypothesis, the mobile phase was initially replaced with an uncharged chaotrope i.e. 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) 50 mM. This did not change the elution order, but increased RT for both NAM and NMN (Fig.  6 ). To eliminate any electrostatic effect, 200 mM NaCl was added to the mobile phase. In these conditions, RT of NAM was the same while NMN RT was slightly shorter, without changing the elution order (Fig.  6E ). This suggested that although electrostatic interactions determine coating repulsion for both NAM and (unexpectedly) NMN, they are not the cause of the reversed elution order, as eliminating them does not restore the theoretical SEC elution order. No further investigations on the matter were performed, as it does not serve the purpose of this study.

figure 6

Separation of nicotinamide mononucleotide (NMN) from (NAM) using uncharged chaotrope mobile phase (50 mM 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP)), isocratic elution, flow: 3 mL/min on PolyHEA column 250 × 9.4 mm, 5 μm, 60-Å. Jasco PU2089 HPLC pump. Detectors: Thermo Velos Pro linear ion trap MS running in ESI + mode, Spray Voltage: 3 kV, Capillary temp: 375 °C, H-ESI-II ion source, connected parallel to MD2015 UV-Vis detector using 1:10 post column flow splitter. Sample volume: 20 μL of standard NMN 10 mM (Sigma Aldrich). Total Ion Current ( A ); 260 nm absorption chromatogram ( B ); 335 Da ions ( C ); 123 Da ions ( D ); The same elution conditions but mobile phase supplemented with 200 mM NaCl, to eliminate the electrostatic repulsion effect, 260 nm chromatogram UV-Vis detector only ( E ).

Although an ultracentrifugation step was performed, that was convenient for small scale processing during the development of the method, for higher scale processing, filtration should be used instead. Moreover, the same filtration method and equipment could be used for both cell separation and lysate clarification. Another advantage of the SEC method presented in this study is that salt molecules (known to be harmful for the mass spectrometry electrospray ionisation source) have a longer RT so the flow can be diverted to waste after elution of the useful fractions, thereby protecting the equipment. Thus, the method applied also performs desalting without the need for any additional processing steps. As previously reported 24 the formic acid from the collected NMN fraction is simply removed by lyophilisation.

Mass spectrometry confirmed NMN peaks identified by the UV-Vis spectrum. Specific MS (Fig.  2 ) and MS/MS spectra (Fig.  3 ), consistent with previously reported data 25 , 26 , 27 were identified in both standard and lysate, increasing certainty of the identity of the obtained product. Considering the molecular diversity of the bacterial lysate, the theoretical possibility to have an impurity with similar size but lower concentration compared to NMN still existed. This type of impurity could hypothetically have the same RT as NMN on the proposed SEC method. As NMN was more abundant, running the MS detector in Full Scan mode could also fail to detect this hypothetical impurity. A typical example is the lack of detection for a 123 m/z peak caused by the fragmentation of NMN in the ionisation source at RT = 6 min during the SEC lysate separation (Fig.  2F ). Although the m/z = 123 signal is present (Fig.  2H ), it is completely eclipsed on the m/z 122.5–123.5 ion chromatogram by the strong NAM peak at RT = 4. Therefore, the product resulting from SEC was evaluated by the second HPLC method based on a completely different separation principle (reversed-phase). During the RP separation, no impurity in the product collected from SEC was observed (Fig.  4B,D,F ). Only the NMN UV absorption spectrum was observed which supported the results of this study that SEC alone separates NMN from the bacterial lysate.

Although the pharmaceutical formulation was beyond the scope of this study, the endotoxin level had to be controlled in case of parenteral administrated drugs. Its concentration was 0.51 EU/mL, below the accepted limit of 1.18 EU/mL. This resulted in a strong suspicion that the detected endotoxin level in the NMN fractions was caused by the HPLC solvents used, which were not labelled as endotoxin free. This hypothesis was supported by the determined endotoxin level in the laboratory water used in the HPLC solvents which was 1.52 EU/mL, three times higher than in the NMN sample. Having a molecular mass greater than 10 kDa, lipopolysaccharides (LPS) resulting from the biotechnological processes are large molecules, which sediment during the ultracentrifugation step. In the less probable situation when they are still present in the clarified lysate, SEC is a known method for their elimination 33 , 34 , and that is the reason for the lower endotoxin level detected in our NMN sample compared with the HPLC solvents used. Evidently, an even lower endotoxin level can be obtained by using endotoxin free solvents. In the case of oral administration, endotoxin contamination is not a concern, as they do not pass through an intestinal barrier 35 .

Growing evidence of anti-ageing and anti-diabetic effects of nicotinamide mononucleotide (NMN) and the current high price tag of this promising molecule were the two key facts motivating us to accomplish this work. The proposed method of NMN purification by Size Exclusion HPLC (SEC) on silica with a covalently attached coating of poly(2-hydroxyethyl aspartamide) (PolyHEA) stationary phase using isocratic elution with denaturing mobile phase (50 mM formic acid) is the first proposed single step chromatographic method for NMN purification from a complex molecular mixture such as a bacterial lysate. The method is simple, patent-free, directly applicable for industrial production with a minimum scale up effort, with bulk material being commercially available to prepare higher capacity columns. It not only eliminates the need for multiple chromatographic steps, but also simplifies the lysate filtration and clarification process, since SEC is not influenced by higher molecular mass compounds which are nonetheless not retained by the column. Therefore, the proposed purification method should substantially contribute to reduction of NMN production costs while also increasing the purity to the level suitable for usage in humans.

NMN Purification Workflow

The process was designed following the classical biotechnological process workflow from the bacterial broth to the pure final product 31 , 36 . The bacterial cells were grown in a 10 L bioreactor and were separated from the media using a 10 L separatory funnel. The cell membranes were disrupted using a sonicator cell disruptor. The cell lysate was clarified by centrifugation and NMN was purified by a single step size exclusion chromatography technique (Fig.  7 ).

figure 7

The schematic diagram for the proposed nicotinamide mononucleotide (NMN) purification process. Bacterial cells are grown in the bioreactor, the media containing bacteria is pumped into a separating funnel and stored at 4 °C. After 24 hours, the sedimented bacterial cells are collected, cell membranes are disrupted by ultrasound on ice, cell debris and macromolecules are separated by ultracentrifugation and discarded, NMN is finally separated by a single step size exclusion chromatography (SEC) on a PolyHEA column eluted with 50 mM formic acid.

Strains, Media, Growth Conditions and Separation of bacterial cells

An overnight culture of E. coli strain BL21(DE3)pLysS genotype E. coli B F–dcm ompT hsdS(rB– m B–) gal λ(DE3) [pLysS Cam r ] [pET28a-hdNadV Km r ] (a gift from Independent Research Association, Bucharest - 012416, Romania), 50 mL, grown in a shaking incubator at 250 rpm was used as inoculum for a 10 L bench-top bioreactor culture. This strain is carrying a plasmid (pET28a-hdNadV, Addgene ID #83362) expressing a gene (nadV) from Haemophilus ducreyi coding for Nampt catalysing the direct production of NMN from nicotinamide (NAM). LB growing media (NaCl 10 g/L, Tryptone 10 g/L, yeast extract 5 g/L) supplemented with 1% NAM and 1% glucose was sterilised in autoclave for 20 minutes at 120 °C. Once the sterilised media cooled down below 60 °C, kanamycin was added to a final concentration of 50 µg/mL. The temperature for bacterial culture was set to 37 °C. The bioreactor was continuously measuring the optical density of the culture at 600 nm (OD600), the stirring motor speed being set to 150 rpm. Once OD600 reached 0.45, the expression of nadV was induced by adding Isopropyl-1-thio-β-D-galactopyranoside (IPTG) to a final concentration of 1 mM. When the measured OD600 of the culture reached 0.75, the media containing bacterial cells was automatically pumped into a 10 L separatory funnel (supplied by Adrian Sistem SRL) and left at 10 °C for 24 h. The funnel tap was then slowly opened, and 100 mL of cells rich media were collected.

Extraction and Clarification

The collected cells were evenly distributed in five 50 mL Falcon Conical Centrifuge Tubes and the cell membranes were disrupted by sonication at 20 kHz on ice in 3 cycles of 30 seconds separated by 30 seconds pause using a sonicator cell disruptor model W185F (Heat Systems-Ultrasonic Inc.). The lysate was ultracentrifuged using the Beckman Optima LE-80 K for 3 hours at 500,000  g at 4 °C. The supernatant was carefully collected and used for NMN purification by High Performance Liquid Chromatography (HPLC).

High Performance Liquid Chromatography (HPLC)

Size exclusion chromatography high performance liquid chromatography (sec-hplc).

Samples of 20 μL were consequently poured on the SEC column.

The Jasco HPLC PU-2089 pump and Rheodyne 20 μL manual injection loop were connected to 250 × 9.4 mm; 5 μm, 60 Å PolyHEA column (silica with a covalently attached coating of poly(2-hydroxyethyl aspartamide) purchased from PolyLC INC (Item# 259HY05006). Post column, a 10:1 ratio flow splitter was used to connect a Jasco UV-Vis Photodiode Array (PDA) MD-2015 on the high flow side, respective of the Heated Electrospray Ionisation (H-ESI-II) from Thermo Scientific Velos Pro Linear Ion Trap Mass Spectrometer on the low flow side. Between the flow splitter and the ionisation source, the flow was passed through the mass spectrometer divert/inject valve as specified in the Thermo Velos Pro user manual. Jasco LC-NETII-ADC controlled by Chrompass 1.8.6.1. software was used to run the LC System while the LTQ Tune Plus 2.7 and Xcalibur 2.2. were used for the mass spectrometry detector and acquisition process. The Jasco LC system was configured to generate the acquisition start signal for the MS to ensure reproducible retention times (RT) were achieved on both data systems. Analog channel 1 from the LC system was connected to the 1 V analogue input of the mass spectrometer. Mass spectrometry grade water (39253 Fluka LC-MS CHROMASOLV) and formic acid (94318 Fluka) were purchased from Sigma-Aldrich as well as other chemicals and reagents, unless otherwise stated. For NMN SEC separation, the isocratic solvent A: 50 mM formic acid was used at a flow rate of 3 mL/min for 20 minutes. The UV-Vis light absorption spectrum (200–640 nm) was continuously recorded. The light absorption chromatogram at 260 nm was recorded by both Chrompass and Xcalibur software.

External standard NMN (Sigma N3501-25MG) solutions were prepared for 5 different NMN concentrations (0.03, 0.16, 0.33, 1.67 and 3.34 g/L), of which 20 µL samples were consecutively eluted by SEC, in the ascending order of concentration. The NMN peak areas of the 260 nm chromatograms were then used to generate the calibration curve by regression 37 for NMN quantification in Chrompass Software (Fig.  1G ).

NMN fractions were collected each time, mixed together, lyophilised and stored at −80 °C.

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

The NMN purified by SEC was hydrated in 200 µL deionised water, 20 µL of the resulting solution was injected in the HPLC sample loop and eluted following the same protocol, previously described.

The Jasco HPLC PU-2089 pump and Rheodyne 20 µL manual injection loop were connected to the Genesis C18 James Column 250 × 4.6 mm; 4 µm; 120 Å column and the Jasco UV-Vis Photodiode Array (PDA) MD-2015 was connected to the column output. The HPLC system was controlled by Jasco LC-NETII-ADC and Chrompass 1.8.6.1. software. A volume of 20 µL of 10 mM NMN (Sigma) standard solution was also injected. Isocratic elution with 10% acetonitrile at a flow rate of 0.9 mL/min for 5 minutes was used. The light absorption spectrum (220–640 nm) was recorded and a 260 nm chromatogram was generated.

Mass spectrometry (MS)

The mass spectrometer connected to the HPLC system as described in the Size Exclusion Chromatography High Performance Liquid Chromatography (SEC-HPLC) section was running in ESI + mode, Spray Voltage: 3 kV, Capillary Temperature was 375 °C. The full m/z range from 70 to 1000 Da was scanned. Prior to sample injection, the mass spectrometer divert valve was switched to the inject position, the MS data acquisition was started from the LTQ Tune software with the “waiting for contact closure” option. Thus, the start acquisition signal was generated by the HPLC injection. After 15 minutes of acquisition, the MS valve was switched to the divert position to protect the instrument from salts. Using Xcalibur software, the total ion current chromatogram and the signal intensity corresponding to m/z between 334.5 and 335.5 Da respective 122.5 and 123.5 Da were extracted from full MS raw data. The 1 V analogue input of the MS was continuously recording a 260 nm absorption data sent by the Jasco MD-2015 plus detector. MS/MS spectra (m/z: 90–340) for NMN containing fractions were obtained by Collision-Induced Dissociation (CID, energy values: 15 and 20) of single charged protonated ion (m/z = 335).

Limulus amoebocyte lysate (LAL) assay

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This study was supported by Independent Research Association, Bucharest - 012416, Romania.

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Marinescu, G.C., Popescu, RG. & Dinischiotu, A. Size Exclusion Chromatography Method for Purification of Nicotinamide Mononucleotide (NMN) from Bacterial Cells. Sci Rep 8 , 4433 (2018). https://doi.org/10.1038/s41598-018-22806-8

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size exclusion chromatography research paper

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Size-exclusion chromatography as a useful tool for the assessment of polymer quality and determination of macromolecular properties

Polymers, macromolecules in nature and bio-polymers like proteins and antibodies are present in our every-day life and play an important role in our economy. They are obviously present as plastic products (e.g., containers, clothing, car tires) or components, e.g., as expedients in pharmaceutical formulations, additives in food and feed, stabilizers in cosmetics, used in construction materials, ceramics and are key in microchip production. Size-exclusion chromatography is the most important characterization technique for macromolecules. This review covers applications, instrumental setup, step-by-step guides for performing experiments and covers theoretical background as well as troubleshooting and tips and tricks.

This text intends to introduce readers to performing successful size-exclusion chromatography (SEC) experiments and covers basic information and guidelines in Section A . After a general introduction in Chapter “ Introduction ” the wide range of SEC applications are summarized to familiarize readers with the technique and its scope (Chapter “ Scope of technique ”). Requirements and experimental details are discussed in Chapter “ Experimental requirements for size-exclusion chromatography ”, which also covers best practices and guidance on avoiding pitfalls. This section is concluded by step-by-step instructions on how to perform successful GPC/SEC (Chapter “ Step-by-step guide to a first SEC analysis ”).

Additional information on SEC advanced methodologies (Chapter “ Optimization of SEC experiments ”) and theoretical background (Chapter “ Theoretical aspects of SEC separations ”) are summarized in Section B for readers which require knowledge about SEC theory and information about adaption and optimization of SEC for specific applications.

Troubleshooting references, additional resources and further reading are covered in Section C at the end of this paper (Chapter “ Further reading and resources ” and following chapters). Supplementary information is available as a slide deck which can be used in teaching this topic (Chapter “ Supplementary information ”).

Introduction

SEC (Size Exclusion Chromatography), GPC (Gel Permeation Chromatography), and GFC (Gel Filtration Chromatography) are all synonyms for the most successfully applied separation technique for the characterization of macromolecules, polysaccharides, (bio)polymers and proteins ( Kilz & Pasch, 2000 ). Although the term SEC is often applied to separations in aqueous solutions and GPC for separations in organic eluents, the same theoretical background, setup, advantages and limitations are valid. However, with respect to the separation mechanism SEC (size exclusion chromatography) is the most descriptive name ( Striegel, Yau, Kirkland, & Bly, 2009 ).

SEC is a liquid chromatography technique which allows to separate molecules based on their size in solution. The size separation step occurs in a column filled with porous particles (stationary phase) and is an entropy-driven process. The sample is dissolved in a solvent, which is also used as a mobile phase in the chromatographic process, and injected onto the column. Larger sizes are excluded from penetrating the pores in the stationary phase while smaller sizes can enter them and thus are retarded on their path through the column. Enthalpic interactions of the sample with the stationary phase surface, as required for HPLC-type separations, have to be strictly avoided in SEC.

SEC allows the measurement of the molecular weights and their distribution of macromolecules. These are in most cases polydisperse, i.e. a physical mixture of different molecules having various chain lengths (molecular weight) in the simplest case. This is the major advantage when selecting an analytical technique for the characterization of macromolecules as competing techniques allow the measurement of bulk properties only ( Kilz, 2006 ).

Although both, SEC and HPLC, are used for chromatographic separation and require similar instrumentation, the separation mechanism, calibration methodologies and result calculation of both techniques is distinctively different. The major differences in the practice of SEC compared to HPLC are outlined in Table 1 .

Important differences between interaction chromatography (HPLC) and size exclusion chromatography (SEC).

In nearly all cases, a macromolecular solution consists of chains with different chain lengths (molecular weights), and, in case of copolymers, additionally of chains with different composition of co-monomers. Each molecule occupies a certain volume in solution, its hydrodynamic volume, that defines the size of the chain in this particular solvent at given physical conditions (temperature, pressure, pH, etc.). If such a polydisperse mixture is injected onto a size-exclusion chromatography column with a matching surface polarity/chemistry (refer to chapter “ SEC column selection ”), a chromatographic separation according to the molecules’ size in solution will occur.

well-defined chain length (molar mass)

specific pentasaccharide sequence (composition)

amount of sulfate groups in the chain (functionality)

Figure 1: Heparin pathway to prevent thrombosis with main active ingredient requirements shown in schematic.

Heparin pathway to prevent thrombosis with main active ingredient requirements shown in schematic.

weight-average molar mass < 8000 g/mol, and

mass fraction > 60% with a molar mass < 8000 g/mol.

Figure 2 shows the results for two Heparin batches which have been obtained by SEC analysis in approx. 90 min. The product sample A (depicted in green) meets both quality requirements containing more than 60% of its mass in molecules with a molar mass of less than 8000 g/mol as compared to sample B (depicted in red), which only meets the M w  < 8000 g/mol requirement by the pharmacopeia for product release.

Figure 2: SEC results of two Heparin batches: sample A passes quality criteria, while sample B fails and is not certified for medical use (courtesy: PSS [Held and Gores, 2010]).

SEC results of two Heparin batches: sample A passes quality criteria, while sample B fails and is not certified for medical use (courtesy: PSS [ Held and Gores, 2010 ]).

The importance of proper heparin quality assurance became obvious when more than 100 people died and several thousand patients fell ill in 2008 after several pharmaceutical companies failed to discover fraudulent heparin materials used in their medical grade production ( Usdin, 2009 ).

Scope of technique

Analyzing macromolecules by SEC has many advantages. First of all, it is a fractionating technique providing access to distribution information as well as to property averages, e.g., all molar mass averages, in a single measurement. Since in most cases nondestructive detectors are used, it is possible to re-collect the sample and/or to investigate the fractions with other analytical methods. Basic laboratory equipment can be used and it is easy to run the samples. Furthermore, a SEC system can be fully adapted to the characterization needs. Different kinds of columns are available enabling goals such as high sample throughput/fast analysis, high information depth/high resolution, saving solvent/green chemistry or high sample loading/small scale clean-up to be met (see Table 2 ). The use of hyphenated techniques ( Kilz & Pasch, 2000 ) and the addition of specialty detectors increase the information content and measurement of different results and distributions simultaneously with just one injection. This short summary explains why SEC is considered an indispensable tool when working with macromolecules.

SEC analysis tasks identifying instrument requirements and typical examples.

Experimental requirements for size-exclusion chromatography

Figure 3 shows schematically a simple lab setup consisting of instrumentation, consumables and computer software for data acquisition and processing.

Figure 3: Schematic setup of SEC instrument including consumables, detection options and MCDS software.

Schematic setup of SEC instrument including consumables, detection options and MCDS software.

Instrumentation

Minimum requirements are a SEC instrument with a solvent delivery pump (with high flow-accuracy), high-pressure injection system (manual or automated), one or more SEC column(s), one or more detector(s) and a computer with a software that allows acquiring, calibrating and analyzing data. Other components such as degasser or column heater/thermostat are optional and depend on the application as well as on the lab environment and conditions. Solvent reservoir, waste bottle and solvent retainer (for the glass bottles) are required to manage eluents safely.

Consumables

High purity solvents (typically > 99%), reference materials for calibration/verification/checkout, syringes, membrane filters.

As size-exclusion, data processing and calibration are unique and distinctively different from conventional HPLC or GC chromatographic data systems (CDS), a macromolecular CDS (MCDS) especially designed for GPC/SEC work is a good investment as it saves time, energy and meets requirements which are special to GPC/SEC. The software is used to control instrumentation, acquire real-time raw data from one or more systems, perform molar mass calibrations, allows to set baselines, integration limits, calculates results and result uncertainties and creates reports.

SEC column selection

The major parameter for column selection is the intended application ( Kilz, 1999 ). A balance of mobile phase polarity in comparison with the polarity of the stationary phase and sample polarity is important for pure SEC separations. In general, users will select their columns according to the mobile phase they need to use. Stationary phase materials can be either silica or polymeric based. Table 3 shows an overview of stationary phases with different polarities typically used in SEC.

Typical stationary phase packings for SEC columns.

After selecting the best matching column material for the analyte, other column parameters have to be taken into account to meet the analysis goals. Here column dimensions, the particle size, and the porosity are of importance.

Column dimensions

Different column dimensions are available. Most common are analytical SEC columns with an inner diameter between 0.7 and 0.8 cm and a length of 25–30 cm. Sixty centimeter columns are still available but rarely used. Preparative columns with the same length but a larger diameter (up to several cm) allow to fractionate larger sample amounts. (Semi)micro columns with approximately the same length and a smaller inner diameter are used with dedicated instrumentation for saving solvent and if only small sample amounts are available.

Particle size

The smaller the particle, the better the chromatographic resolution as dispersion effects are minimized ( Kilz, 2006 ). However, high molar masses and high viscous solvents require larger particle sizes because the injection solution cannot be diluted by the eluent flow through the column. So there is an optimum particle size depending on the application ( Kilz, 2006 ; Striegel et al., 2009 ).

Another important parameter for column selection is the proper choice of sorbent porosity ( Kilz, 2006 ). The molar mass range of the samples to be investigated determines the column porosity. The larger the pores, the higher molar mass samples can be characterized. Unfortunately, there is no general nomenclature, which will allow easy selection of column pore sizes. Each manufacturer has its own system for pore size designation. The easiest method to find out which columns will be useful for a selected task uses the calibration curve which every manufacturer shows in their literature.

In general, SEC columns can be either single porosity columns with narrow pore size distribution or linear (also called mixed-bed) or multipore columns with a very broad pore size distribution. SEC separation capacity is limited by the available pore volume and depends on sorbent type, column dimensions and the slope of the calibration curve. The highest selectivity for a separation is determined by the lowest slope of the calibration curve.

For single porosity columns the separation capacity is concentrated in a narrow molar mass range. This yields a calibration curve with a flat or shallow slope in this region. Therefore, single porosity columns have a limited molar mass separation range, but a high resolution in that range. In contrast to that, columns with a broad pore size distribution provide a larger separation range and the calibration curve has a steeper slope and therefore less resolution.

Often linear or mixed-bed columns are either used in QC for fast screening experiments or to identify the molar mass range of a sample, so that it can be investigated on a matching single porosity column bank with higher precision.

SEC detector selection

There is a wide range of chromatography detectors available on the market. However, not all are useful in macromolecular analysis and several “specialty” detectors are specific to macromolecular analytes. An in-depth discussion of detector technology for SEC use can be found in References ( Kilz & Pasch, 2000 ; Striegel et al., 2009 ).

As each chromatography technique requires the online determination of concentrations of each separated (analytical) fraction such detectors are required in every instrumental setup.

Concentration detectors

Refractive index detectors (rid).

The RID measures the change in refractive index of the column effluent passing through the flow cell compared to that of the pure eluent stored in the reference cell.

This is the most useful and generally applicable concentration detector in SEC as it detects most analytes independent of, e.g., solvent and wavelength (as in the case of an ultraviolet spectroscopy detector (UVD)). Only isorefractive samples cannot be detected. An example for this is poly(dimethylsiloxane) in tetrahydrofurane (THF). Such samples should be analyzed, e.g., in toluene to ensure proper concentration determination.

RID detectors are primarily used to measure concentration profiles. In addition, they are used to measure the fraction concentration when working with online light scattering detectors, viscometers or online mass spectrometry.

In combination with other concentration detectors they are used to measure the comonomer distribution and molar mass in copolymers or end group distributions.

Disadvantages of this detector are its low sensitivity, relatively long times to stabilize, their tendency to drift (especially with poor thermostatization and in solvent mixtures) and their large cell volume.

Variable wavelength (UVD) and diode-array (DAD) detectors

They measure the UV adsorption at a fixed (user-selectable) wavelength for samples with chromophores. Since SEC is used to measure the molar masses and the distribution, it is sufficient to measure at one or two fixed wavelengths where the sample shows absorption. Spectra from diode array detectors (typically used for substance identification in HPLC) are only rarely needed, e.g., for the analysis and identification of oligomers with special properties.

UVD detectors are used to measure concentration profiles and (if used alone) molar masses based on a calibration curve. In addition they are used to measure the fraction concentration required when working with online light scattering detectors, viscometers or mass spectrometers. They are the most common detectors for protein characterization.

UV detectors possess small cell volumes, are easy to use and have a good sensitivity (compared to a RID). A disadvantage of this detector is that it can only be applied for a limited number of polymeric samples due to missing chromophores (e.g., in PDMS, PVC, PE, PEG/PEO) and potentially strong changes of response factors with minor chemical or oligomer molar mass variation.

Evaporative light-scattering detector (ELSD)

This detector destroys (evaporates) the mobile phase to create a particle stream with the number of particles changing with the analyte concentration. However, the relationship between sample concentration and response (peak area) is not linear and therefore this detector is not suitable for quantitative analysis. The main advantage of this detector is the high sensitivity, suitability for solvent gradients and that it can detect samples which do not possess chromophores.

This detector cannot be used with high flow rates, samples which evaporate and eluents which contain non-volatile components (e.g., salts).

Note that this is not a detector to measure molar masses (weight-average molecular weight) in solution, since it uses light scattering to determine the number of particles in non-condensed phase.

Non-concentration detectors

Online light scattering detectors.

They measure at one or more fixed detection angle(s) the time-averaged intensity of light scattered by macromolecules in solution. Low-angle laser light scattering (LALLS) detectors measure scattered light intensity at 5–7° and right-angle laser light scattering (RALLS) detectors at 90°. Multiangle laser light scattering (MALLS) detectors measure light intensity at multiple angles simultaneously. They are used to determine absolute molar masses for homopolymers and proteins and polymer structures in solution (MALLS only).

Online viscosity detectors

Online viscometers come in a range of different configurations (single, dual, or four-capillary type viscometers with a symmetrical or asymmetrical bridge). The most suitable type is a 4-capillary bridge viscosity detector which combines high sensitivity with flow independence.

They measure the pressure difference between a sample path and a reference path filled with pure solvent. Viscometers are used to measure specific and intrinsic viscosity, molar masses based on Benoit’s universal calibration approach ( Grubisic, Rempp, & Benoit, 1967 ) and Mark-Houwink coefficients.

Mass spectrometry detectors

Different mass spectrometric methods have been used in macromolecular analysis. They are used to determine absolute molar masses for homopolymers and copolymers and to detect polymer structures. Matrix Assisted Laser Desorption Ionization–Time of Flight (MALDI-ToF) and Electrospray Ionization (ESI) are the most common instruments used in combination with SEC. A recent application summary can be found in ( Barner-Kowollik, 2011 ).

IR detectors

Online detection with an IR detector is mainly used in high temperature SEC (HT SEC) for the characterization of polyolefins. FTIR signals provide information, e.g., on short chain branching, if the ratio of different wavelengths is compared. For many other SEC applications online IR detection cannot be used due to the fact that the typical solvents absorb in the same region as the investigated polymers. Far more important are offline techniques ( Kilz & Pasch, 2000 ). The SEC effluent is directed to a heated nozzle for evaporation of the solvent followed by sample deposition on a germanium disc. The disc is then scanned in an offline step in standard FTIR spectrometers. This elegant technique allows to separate and to detect without the influence of the solvent and is often used in additive analysis to identify unknowns, e.g., in master batches.

Modern SEC instruments in R&D are today often equipped with 3 or 4 detectors in series. However, this does not minimize the user input required and the knowledge of each method applied (SEC, light scattering, viscometry) is indispensable. Additional system parameters, such as the inter detector delay, need to be determined carefully as they influence the results ( Held & Kilz, 2009 ). In addition band broadening due to use of several detectors can be a problem ( Gaborieau, Gilbert, Gray-Weale, Hernandez, & Castignolles, 2007 ; Mader and Schnöll-Bitai, 2005 ; Meira, Netopilík, Potschka, Schnöll-Bitai, & Vega, 2007 ).

Best practices and avoiding experimental pitfalls

Despite general knowledge about chromatography instrumentation, successful macromolecular characterization by SEC/GPC/GPC techniques require special attention to instrument modules, sample preparation, eluent preparation and column use. The following chapters present best practices and how to avoid common pitfalls and oversights. Additional information and details on working with advanced detection systems can be found in ( PSS Polymer Standards Service, 2019 ).

Instrument readiness testing

Table 4 summarizes important experimental component features and provides a test description with pass criteria for successful operation of SEC instrumentation ( PSS Polymer Standards Service, 2019 ).

It also lists items which are often overlooked and can make lab life difficult or render analytical results ambiguous.

Hardware readiness test.

Sample preparation

– Allow samples to be dissolved on a molecular level in order to prevent aggregation/agglomeration and thus abnormal chromatograms.

– Ensure eluent and sample compatibility with installed separation columns.

– Verify stability (degradation, reactivity with samples/sample constituents, … ).

– Consider environmental, health and safety aspects (e.g., toxicity, elevated temperature, etc.).

– In order to minimize so-called system peaks, especially for RID, use a portion of solvent directly from the eluent reservoir for sample preparation and minimize injection volume (if possible).

– Avoid high shear, stirring, sonication during dissolution.

The optimal sample concentration depends on molecular weight and sample polydispersity (local concentration in the column during elution); refer to Table 5 for guidance.

Recommended sample related parameters.

a sample concentration also depends on sample polydispersity PDI (higher PDI allows higher conc.). b dissolution time depends also on temperature and crystallinity of the sample.

Recommended sample concentrations and dissolution times for typical samples analyzed on conventional SEC columns (typical dimension: 300 × 8 mm ID) in good solvents are presented in Table 5 .

Sample filtration:

– Filter off insoluble residues through 0.2–1.0 μm membrane filters (e.g., use disposable syringes with suitable filters, consider solvent and sample compatibility).

– Some samples (e.g., natural rubber, native starch samples, … ) might be difficult to filter. Then, centrifugation is the method of choice.

Eluent recommendations

– eluent and solvent for sample preparation must be filtered through <10 µm membrane filters after additives, salts, etc. have been dissolved

– eluent temperature should be < 30 °C below its boiling point

– eluent must be free of visible bubbles before being fed into the pump

– replace eluent regularly (typically every two weeks)

– eluent should be kept in brown glass bottles and not be exposed to direct sunlight, aqueous mobile phases need additives (e.g., 0.05 M sodium azide) to prevent algae growth

– eluent bottle must be capped with ability for atmospheric pressure exchange

– system effluent must not be fed back to the eluent reservoir on a regular basis

– flush all new tubing with eluent before connecting to a module/column

– flush columns with eluent (approx. 3x column volume) before connecting to a detector

– eluents containing salts are not suitable for storing columns and should not be left in in detectors or in systems without mobile phase flow

Step-by-step guide to a first SEC analysis

This guided SEC analysis assumes that the SEC system as outlined in chapter “ Experimental requirements for size-exclusion chromatography ” is equipped with a (minimum) RID detector and operating in THF as the eluent and a SEC column packed with styrene-divinylbenzene (SDV) packing material. Each injection requires approx. 15 mL of eluent and sample elution will take 15 min to complete (at a pump flow rate of 1 mL/min).

Ensure that the SEC system is operational and ready to run (see chapter “ SEC detector selection ” and Reference ( PSS Polymer Standards Service, 2019 ) for details).

Attach a linear (mixed bed) analytical column (with approx. molar mass range 1–3000 kg/mol) to the SEC system. Start the pump to deliver eluent through the column for a minimum of 4 column volumes (approximately 1 h at 1.0 mL/min). After column conditioning is complete, stop the pump flow, connect the column outlet to the detector(s) and start the pump up again.

Dissolve polystyrene molar mass calibration standards for the calibration of your SEC system by adding the required eluent volume from the reservoir bottle to each of the calibration vials (refer to Table 5 for details). Vials should sit a minimum of 2 h on the lab bench without agitation (vortex, ultrasound, etc.). The most easy-to-use reference standards are pre-mixed so-called ReadyCal standards which just require mobile phase addition to get ready ( PSS Polymer Standards Service, 2016 ).

Start your chromatography data system (preferably a dedicated (optimized) SEC MCDS software package ( Kilz, 2019 ) and record the baseline.

Perform a column plate count and asymmetry test as described in the column user documentation or on the certificate of analysis.

Perform a test injection with one of the calibration standard solutions and determine the detector signal drift (which should be < 5% of the maximum peak height) and signal noise (<2% of peak maximum height). Repeat injection if pass criteria are not met (see Table 4 for remedies). Continue to step 7 if signal drift and baseline noise pass test criteria. If the test fails, replace the column with approximately 3 m of new stainless steel capillary (0.1 mm internal diameter) and rerun the test. If the test now passes, replace the SEC column with a new one. If the signal test still fails, follow the troubleshooting hints in the detector user documentation.

Inject 20 µL of each of the calibration standard solutions, create a molar mass calibration table, and graph in the chromatography data system as shown in Figure 4 .

Save the calibration and assign it to the current sample sequence.

Create or modify an existing data acquisition method with the calibration established in step 8.

Prepare a broad Polystyrene reference material (e.g., available from www.pss-shop.com ) or a commercial polystyrene sample (e.g., Styrofoam, plastic cup) by dissolving the sample in the eluent over night without agitation. After >12 h swirl the sample solution slightly and filter it into a glass vial/bottle.

Load the data acquisition method prepared in step 9 and inject 50 µL of sample solution in triplicate. Process all 3 injections, calculate the molar mass results and molar mass distribution. Molar mass averages of all three repeat injections should deviate <5% for M w and <10% for M n and M z . Overlay the 3 chromatograms (elugrams) and molar mass distribution curves for review as shown in Figure 5 .

Figure 4: a) SEC chromatogram of a Polystyrene PSS ReadyCal reference material with UVD (red) and RID (green) detector traces.

a) SEC chromatogram of a Polystyrene PSS ReadyCal reference material with UVD (red) and RID (green) detector traces.

Figure 4: b) Molar mass calibration graph (top) and numeric table with details for each of the 12 calibration reference materials.

b) Molar mass calibration graph (top) and numeric table with details for each of the 12 calibration reference materials.

Figure 5: Triplicate SEC analysis of broad Polystyrene CRM (PSS-BAM P002): molar mass distributions and results overlay shown in top graph, raw signal overlay shown below.

Triplicate SEC analysis of broad Polystyrene CRM (PSS-BAM P002): molar mass distributions and results overlay shown in top graph, raw signal overlay shown below.

Optimization of SEC experiments

SEC separations require interaction-free diffusion of the sample molecules into and out of the pores of the stationary phase. In general, this goal is easier to achieve in organic eluents than in aqueous solutions. In aqueous mobile phases more parameters (e.g., type of salt, salt concentration, pH, addition of organic modifier, concentration of co-solvent) have to be adjusted correctly. In addition, due to the presence of charged functional groups, hydrophobic, and/or hydrophilic regions in the molecule, water soluble macromolecules have more possibilities to interfere with the stationary phase.

A proper SEC experiment has to be balanced with respect to polarities. In order to obtain a true and pure SEC separation, the polarity of stationary phase (column material), the polarity of eluent and the polarity of sample have to be matched. This is visualized by the magic triangle (see Figure 6 ). Dominance of size separation is only maintained in the center of the triangle (bright area). where the overall system is balanced. Otherwise specific interactions will occur, which will overlay with the normal SEC elution behavior.

Figure 6: Balancing the polarities of the phase system in SEC applications for interaction-free separations; optimization of sample, eluent and column for a) organic eluents and b) aqueous mobile phases (courtesy: PSS Polymer Standards Service, pss-polymer.com).

Balancing the polarities of the phase system in SEC applications for interaction-free separations; optimization of sample, eluent and column for a) organic eluents and b) aqueous mobile phases (courtesy: PSS Polymer Standards Service, pss-polymer.com ).

SEC method optimization

To increase the resolution and/or the separation range a very simple approach can be applied. Instead of just using one column, multiple columns are combined to a column combination or a column bank; 2 to 4 columns (plus a guard column) are typical in SEC. A column combination or column bank provides more available pore volume for more efficient separations. If two columns with the same pore sizes (single porosity or linear/mixed bed/multipore) are combined, the slope of the calibration curve will be smaller and the resolution increases by a factor of 1.4 ( Kilz, 2006 ). However, the separation time increases by a factor of 2 (refer to Figure 7 for a visual impression). If columns with different porosities are combined the molar mass separation range increases ( Kilz, 2006 ).

Figure 7: Comparison of SEC resolution enhancement of myoglobin unimer-dimer pairs by increasing the column length by a factor of 2.

Comparison of SEC resolution enhancement of myoglobin unimer-dimer pairs by increasing the column length by a factor of 2.

Figures 8 and 9 show a comparison of the same sample mixture analyzed on two different column banks. In Figure 8 the columns are optimized for the characterization of low molar masses while the column bank in Figure 9 is optimized for the separation of medium molar masses. This example illustrates also the influence of the slope of the calibration curve on the resolution, as well as the difficulty for inter-laboratory comparison of chromatograms: the look of chromatograms or the raw data depends of the columns used. Therefore it is always recommended to compare the molar mass distribution instead of the chromatograms or raw data.

Figure 8: Separation of a poly(styrene) standards cocktail on a SEC column bank optimized for oligomer separation by combining narrow pore-size columns; the flat calibration curve indicates best resolution at low molar mass.

Separation of a poly(styrene) standards cocktail on a SEC column bank optimized for oligomer separation by combining narrow pore-size columns; the flat calibration curve indicates best resolution at low molar mass.

Figure 9: Separation of the same poly(styrene) standards cocktail as in previous Figure on a SEC column bank optimized for medium molar masses. The lower resolution in the low molar mass region results in a single peak instead of multiple peaks for each single oligomer.

Separation of the same poly(styrene) standards cocktail as in previous Figure on a SEC column bank optimized for medium molar masses. The lower resolution in the low molar mass region results in a single peak instead of multiple peaks for each single oligomer .

Disadvantages of column banks are that price, pressure, analysis time and eluent consumption increase. An increased pressure might result in the need to reduce the flow-rate and/or to increase the temperature to have better chromatographic conditions, especially for high molar mass macromolecules. In addition, there is the potential danger of porosity mismatch for all column types, linear/mixed bed or single porosity alike. Porosity mismatch often shows itself in peak shoulders which might be misinterpreted as better resolution, but are artifacts of a column bank due to nonmatching porosities. This phenomenon can also be observed if non-matching porosities are mixed in one linear/mixed bed column to provide a wide linear separation range.

Method optimization with respect to a better resolution includes also to adjust all parameters that improve the mass transfer. The following parameters can be used to optimize the separation:

Theoretical plate height and column permeability decrease with the particle diameter ( Kilz, 2006 ). Smaller particle size columns provide therefore a better resolution. This concept, that led to the development of UHPLC, can also be adapted to SEC taking some peculiarities into account. Figure 10 shows a comparison of a protein mixture measured on the same column material with different particle sizes. The mass transfer for the 5 µm material is much better resulting in an increased resolution. Therefore, if the molar mass and rigidness of the macromolecules permit and no shear degradation occurs, the higher prices for small particle columns are a good investment in higher resolution.

Figure 10: Influence of particle size of column packing material on the resolution of a protein mixture under otherwise identical conditions.

Influence of particle size of column packing material on the resolution of a protein mixture under otherwise identical conditions.

The general rule of thumb is that oligomers in low viscous solvents and proteins allow columns packed with 3 µm particles, for medium molar masses 5–10 µm particles are recommended and for high molar masses and high viscous solvents 10–20 µm particles sizes are used as column packing materials.

1 mL/min flow-rate is often applied for analytical SEC columns with an inner diameter between 7 and 8 mm as the flow-rate with the best compromise between resolution and analysis time. Especially for higher molar masses a decrease of the flow-rate results in a higher resolution. Columns with larger inner diameter are best operated with higher flow-rates while columns with smaller inner diameter are used with lower flow-rates to maintain the same linear flow velocity.

Temperature

A temperature increase will in general also result in a better resolution due to the enhanced mass transfer. However, this is not applicable for all macromolecules. For example, polyethylene glycol (PEG) in aqueous solution shows a better resolution at lower temperatures.

High temperature SEC systems, where the complete system is heated, is needed for macromolecules that are only soluble at elevated temperatures, e.g., polyethylene (PE) or polypropylene (PP).

Table 6 summarizes various ways to optimize SEC separations by proper selection and combination of SEC columns.

Optimization of SEC separations.

Optimizing detection in SEC

At least one detector in SEC is required to detect the eluting sample. In many cases, SEC instruments for research are fitted with 3 or 4 detectors in series. However, advanced detection hardware capabilities come at a price as they require additional care and operator knowledge. Moreover, additional instrument parameters, e.g., the inter detector delay volumes, are required as they may bias the results significantly ( Held & Kilz, 2009 ). Special attention has to be taken on band broadening caused by the multiple cell volumes and connection capillaries when several detectors are incorporated into a SEC instrument ( Gaborieau et al., 2007 ; Mader and Schnöll-Bitai, 2005 ; Meira et al., 2007 ).

The molar mass distribution is then derived from the measured concentration of the separated fraction, its molar mass (from a calibration curve or measured directly using additional detectors as light scattering detectors or mass spectrometers), and the slope of the calibration curve.

The requirements for detectors in SEC are the same as for detectors in other methods: first of all, the detector must be able to detect the sample in the desired application, while the ability to detect a broad range of samples is a definite plus. Additional requirements, e.g., sensitivity, detection limit, linearity, baseline drift, noise, cell volume, and ease-of-use have to be taken into account. As detector design is discussed in many chromatography books (e.g., Kilz & Pasch, 2000 ; Striegel et al., 2009 ; Meyer 2010 ), this text will focus on the applicability and usability of detectors in polymer analysis.

In contrast to HPLC, the combined parallel or in series use of detectors with different principles is one of the major advantages in modern SEC experiments. It allows access to more detailed sample information, sometimes to absolute molar masses, sometimes to other types of distributions, that can be present in complex polymeric samples (e.g., chemical composition distribution, end group distribution, structural distributions, etc.). Table 7 presents an overview of typical detector combinations used to investigate specific sample properties. Additional information can be found in review papers ( Kilz & Pasch, 2000 ; Striegel et al., 2009 ) and in the Supplementary Information .

Summary of SEC applications with different detector combinations.

Theoretical aspects of SEC separations

The basic principle of chromatography separation can be described by simple thermodynamic principles applying the thermodynamic distribution coefficient, K :

with a activity (concentration) of the molecule in the stationary phase (indexed s ) and the mobile phase (indexed m ).

Δ G free energy change between the species in the stationary phase and the mobile phase.

R universal Gas constant.

T absolute temperature.

Δ H enthalpy difference between the species in the stationary phase and the mobile phase.

Δ S entropy difference between species in the stationary phase and the mobile phase.

In SEC separations, the enthalpic contribution, Δ H , to the free energy term is negligible, assuming no energetic interaction between analyte and sorbent as expressed by the size-exclusion distribution coefficient, K SEC :

Δ S entropy loss when a molecule enters the pore of the stationary phase.

In the HPLC case of non-steric interaction of the molecule with the stationary phase, the retention can be described by the enthalpic term alone, as expressed by the HPLC distribution coefficient, K HPLC :

Δ H enthalpy change when a molecule is adsorbed by the stationary phase.

Equations (2) and (3 ) describe the two ideal extremes of chromatography (SEC and HPLC), when there is no contribution of entropy or enthalpy, respectively.

Calculation of molar mass averages

SEC separates based on the hydrodynamic volume and the molar mass information is only available when a correlation between molar mass and elution volume has been established by a calibration or absolute detection by molar mass sensitive detectors.

The calculation of the molecular weight averages uses the so-called slice method ( Held & Kilz, 2009 ; Kilz, 2006 ; Schröder, Müller, & Arndt, 1998 ). The eluted concentration profile is cut into equidistant volume slices and the elution volume domain is transformed to the molar mass domain.

The molecular weight averages are defined and calculated by:

with h the signal height and M the molar mass of the slice (analytical fraction) i .

μ represents the moment of the distribution function and are a more general method to calculate different averages of distributions. The molecular weight averages can then be calculated from the moments, µ , of the molar mass distribution, w(M) , as described above ( Held & Kilz, 2009 ; International Organization for Standardization, 2008 ; Schröder et al., 1998 ):

The width of the molar mass distribution can be described by the polydispersity, D , also called polydispersity index, PDI:

However, molar mass averages are reduced information only and do not describe a polydisperse sample comprehensively. The macroscopic properties of macromolecules can better be derived from their molar mass distribution, w ( M ). Two samples can have the same molar mass averages but still have very different molar mass distributions and therefore macroscopic properties. It is possible to derive the molar mass averages from the molar mass distribution but not vice versa.

The molar mass distribution can be calculated from the signal heights, h (V).

The differential distribution, w ( M ), of the molar mass M is defined as

where: dm / dM is the mass fraction of polymer in a dM interval

By simple transformations, w ( M ) can be expressed by quantities measured by SEC instrumentation directly:

with: h ( V ) detector signal with elution volume, V , as observed in the chromatogram.

σ (V) slope of the calibration curve.

M ( V ) molar mass change with elution volume, V .

The correction with the slope of the calibration curve is necessary, because the data recording is linear in the time domain while the molar mass does not increase linearly due to the separation process (calibration influence). This means, that the number of polymer chains with the same concentration on the high molecular weight part of the chromatogram is much smaller than on the low molecular weight part. Only with strictly linear calibration curves, a behavior observed only for a very limited number of setups, the correction is not needed.

Fundamentals of SEC calibration

The primary information obtained from SEC is not the molar mass, but the apparent concentration at an elution volume. Only by matching SEC calibration and the concentration profile from the concentration detector can the molar mass averages and the molar mass distribution be obtained. SEC is therefore a relative method if no absolute detection is employed ( Striegel et al., 2009 ). The SEC calibration is based on assigning a molar mass to an elution volume (calibration of x -axis). This is in contrast to HPLC, where the detector response (signal intensity, peak area) is calibrated and assigned to a concentration (calibration of y -axis).

– narrow molar mass distribution

– broad molar mass distribution

– use of an online viscometer and a concentration detector and universal calibration of the SEC system,

– use of a light scattering and a concentration detector,

– use of a mass spectrometer and a concentration detector.

SEC calibration with reference materials with narrow molar mass distribution is by far the most commonly used and most accurate method for calibration ( International Organization for Standardization, 2008 ; Schröder et al., 1998 ). The standards come with certificates showing at least the molar mass averages. They are used for conventional calibration or for universal calibration with or without an online viscometer. The calibration curve is created by measuring the elution volumes of the reference materials and by plotting them versus the logarithm of the molar masses (in general the molar mass at the peak maximum). Then a fit function, that describes the shape of the calibration curve, has to be chosen. Unfortunately, there is no general fit function that can be used for all columns/column banks, but users have to select a proper fit function based on multiple criteria.

Most calibration curves, often even the ones for linear or mixed-bed columns, have a sigmoidal shape. This is in agreement with the fundamental separation characteristics and in contrast to other calibrations in chromatography, where linear calibration curves for the peak area plotted versus the concentration are obtained.

A SEC calibration curve can be divided into three distinct regions, as shown in Figure 11 . Domain I represents the exclusion limit of the column(s), where no separation occurs as species larger than the largest pores in the column packing elute at this position in the chromatogram. Domain II is the optimum size separation range for macromolecular characterization. Molecules are separated according to their hydrodynamic volume in solution. Large molecules with high molar masses elute first; molecules with lower molar masses and smaller hydrodynamic volumes elute later. Domain III represents sample interaction with the stationary phase (HPLC mode). This limit is called total penetration volume, where all molecules elute that are smaller than the smallest pores in the column packing.

Figure 11: Generalized shape of SEC calibration curves covering the complete separation range (V0 represents complete exclusion from pores, Vt represents total penetration into pores).

Generalized shape of SEC calibration curves covering the complete separation range ( V 0 represents complete exclusion from pores, V t represents total penetration into pores).

In most cases SEC fit functions are based on polynomial functions with a degree of 3 (cubic) or higher (4–7). If a linear (mixed-bed) SEC column is used in the separation, a linear calibration fit can be applied. There are also special dedicated fit functions (e.g., PSS calibration functions) available that are based on polynomial functions, but optimized for the SEC separation behavior to avoid typical pitfalls (refer to the calibration curve shown in Figure 11 which must be represented by the user-selected mathematical equation).

Three decision criteria can help to decide if the proper function has been selected: These are the regression coefficient, R 2 , the deviation of the calibration point from the fitted value (e.g., average deviation), and the slope of the calibration curve.

Table 8 illustrates this decision making process. It shows the regression coefficients and the average deviation for all data points for identical calibration data fitted with different functions. It is obvious that the regression coefficient alone is not a proper parameter to select the best fit function. Large average deviations are observed even for a regression coefficient very close to unity. If the SEC software provides the regression coefficient as the only selection criterion, a value of R 2  > 0.999 should be achieved.

Influence of the calibration fit function on the regression coefficient and the average relative deviation.

In addition, this table shows that when polynomial functions with a higher degree are selected, the regression coefficient and the average deviation become smaller. However, it is not physically meaningful to use the function with the highest degree, despite that this will always generate the lowest average deviation. More important than small deviations is that the shape of the calibration curve is in general agreement with the separation mechanism. A good measure for a physically meaningful fit is the first derivative of the calibration curve, the slope of the calibration curve.

Figure 12 shows an ideal first derivative for a calibration curve. The slope is constant for the optimum separation range and changes only close to the exclusion limit and the total penetration volume. If a higher polynomial fit function (e.g., seventh degree used in Figure 13 ) is chosen, local maxima and minima, lacking any physical significance, will appear. Over-fitting should be avoided, since it can produce artifacts in the molar mass distribution (e.g., shoulders), that are not related to the sample characteristics.

Figure 12: Example of a good SEC calibration with small deviations and continuous first derivative which covers the complete column pore volume.

Example of a good SEC calibration with small deviations and continuous first derivative which covers the complete column pore volume.

Figure 13: Poor SEC calibration fit (same data points as in Figure 12) with small deviations but discontinuous first derivative which will lead to artifacts (e.g., shoulders) in the molar mass distribution.

Poor SEC calibration fit (same data points as in Figure 12 ) with small deviations but discontinuous first derivative which will lead to artifacts (e.g., shoulders) in the molar mass distribution.

Therefore the optimum fit function is the one with the lowest deviations that still has a constant slope without maxima or minima.

However, one of the major limitations in SEC is that the separation is based on the hydrodynamic volume. This does not only depend on the molar mass of the molecule but also on its chemical nature and topology. Therefore a calibration curve created from reference materials is strictly only valid for samples with the same chemistry and topology. For other samples apparent molar masses will be obtained. It is still possible to compare the samples, but it is not possible to measure accurate molar masses.

There are many different types of reference materials available to create the matching calibration curve for many samples. In addition universal calibration with Mark-Houwink coefficients and broad calibration methods are available.

Online viscometers can be used to measure a universal calibration curve ( Grubisic, Rempp, & Benoit, 1967 ). Here the logarithm of the hydrodynamic volume, the molar mass multiplied by the intrinsic viscosity, is plotted against the elution volume. Universal calibration curves are valid for all types of polymers and copolymers independent on the topology. For creating a universal curve and choosing a fit function the same rules apply as for a conventional calibration curve. The only difference is that the intrinsic viscosity measured using the viscosity detector is additionally used.

Molar mass sensitive detectors such as online light scattering detectors and MS detectors allow to measure the molar mass at every elution volume directly.

The calibration options mentioned in b) and c) are beyond this introductory text; more information can be found in ( Kilz & Pasch, 2000 ; Striegel et al., 2009 ).

Further reading and resources

It is impossible to cover all aspects of size-exclusion chromatography, its application to various macromolecules and how to troubleshoot instrumental or separation method in a single paper. Instead, this chapter summarizes important target industries with their respective SEC applications and lists resources for further reading. Relevant resources listed below are collected to the authors’ best knowledge, but might change as Internet offerings are very dynamic.

Markets and applications for size-exclusion chromatography

Table 9 summarizes important markets for macromolecular products and SEC application fields (non-exclusive listing; many others are known). Application resources are presented in chapter “ Where to find SEC applications? ”.

Commercial use of SEC analysis and important application fields.

Where to find SEC applications?

Internet search is certainly a first resources especially if the reader is working on a current topic probably covered in primary literature. However, do not search for the term SEC but for GPC. Otherwise, search results for SEC will most certainly obstruct relevant information behind a huge number of references to publications related to the United States “Securities and Exchange Commission” also known as “SEC”.

A good resource for meaningful application notes and compendia are the major vendors for SEC instrumentation and consumables listed in Table 10 .

Major chromatography vendors, their SEC product, and service offering.

How to find SEC troubleshooting information

The first stop for finding valuable information about a product is the product documentation itself. In most cases the user documentation will cover installation and use and also contain general chromatography troubleshooting information which might not address a specific SEC aspect, however. Most vendors listed in Table 10 will offer specific guidance on their SEC products.

Separation Science Lab Journal offers a series of “GPC/SEC Good Practice & Troubleshooting Tutorials” ( https://learning.sepscience.com/form/gpc-sec-good-practice-troubleshooting-tutorials ).

SEC tips and tricks for meaningful results

Most vendors listed in Table 10 will offer comprehensive documentation and optimization for their SEC products.

Practical knowledge and how to make SEC painless and avoid non-obvious traps can be found in the following resources:

LCGC and PSS: collection of GPC/SEC tips&tricks written by PSS scientists

https://www.pss-polymer.com/en/support/librarypss-publications/gpcsec-tipstricks.html .

Chromatography online: collection of GPC/SEC advice written by PSS scientists

https://www.chromatographyonline.com/search?searchTerm=sec%20tips .

LaborPraxis and PSS: collection of more than 70 GPC/SEC tips written by PSS practitioners (in German)

https://www.pss-polymer.com/de/support/bibliothek-pss-publikationen/gpc-tipps-und-tricks-deutsch.html .

PSS tips on software best practices as well as tips and tricks: https://www.pss-polymer.com/en/support/software-support/wingpc-newsletter.html .

Conclusions

SEC characterization of macromolecular products with various compositions and architectures deliver deep insight into molecular design and allow the establishment of structure–property–function correlations. The results are robust, repeatable and dependable and are indispensable in product control and QA release when SEC specific procedures are adhered to as outlines in this paper.

Supplementary information

Lecture notes on SEC from the Characterization Workshop during the IUPAC MACRO Congress 2016 in Turkey are available as supplementary information. The slides focus on size-exclusion methodology and expand the scope of this introductory text for advanced detection and separation techniques. Additional applications demonstrate the use of these techniques. PowerPoint slides may be obtained by the author for educational use.

Abbreviations and acronyms

butylated hydroxytoluene (2,6-Di-tert-butyl-4-methylphenol)

chemical composition distribution

diode-array detector

dimethyl acetamide

dimethyl formamide

dimethyl sulfoxide

evaporative light-scattering detector

functional-type distribution

Fourier-transform infrared spectroscopy

gas chromatography

gel filtration chromatography

gel permeation chromatography

high performance liquid chromatography

distribution coefficient

liquid chromatography

molecular architecture distribution

matrix-assisted laser-desorption ionization mass spectrometry

molar mass distribution

mass spectrometry

N -methyl pyrrolidone

nuclear magnetic resonance

poly(acrylic acid)

poly(butadiene)

poly(carbonate)

poly(dimethyl siloxane)

poly(ethylene)

poly(ethylene glycol)

poly(ethylene oxide)

poly(ethylene terephthalate)

poly(iso-butylene)

poly(lactic acid)

poly(methyl methacrylate)

poly(oxymethylene)

poly(propylene)

poly(styrene)

polyurethane

poly(vinyl chloride)

quality assurance

quality control

refractive index detector

size-exclusion chromatography

trichloromethane

tetrahydrofurane

ultra-violet detector

Acknowledgments

The authors are indebted to Felix Ho, Associate Professor at Uppsala University, for his support and invaluable advice to amend the manuscript to the audience of Chemistry Teacher International. We also want to thank our co-workers at PSS for their support, their work and exciting discussions.

Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

Research funding: None declared.

Conflict of interest statement: The authors declare no conflicts of interest regarding this article.

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Size-Exclusion Chromatography: A Twenty-First Century Perspective

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Now in its sixth decade, size-exclusion chromatography (SEC) remains the premier method by which to determine the molar mass averages and distributions of natural and synthetic macromolecules. Aided by its coupling to a variety and multiplicity of detectors, it has also shown its ability to characterize a host of other physicochemical properties, such as branching, chemical, and sequence length heterogeneity size distribution; chain rigidity; fractal dimension and its change as a function of molar mass; etc. SEC is also an integral part of most macromolecular two-dimensional separations, providing a second-dimension size-based technique for determining the molar mass of the components separated in the first dimension according to chemical composition, thus yielding the combined chemical composition and molar mass distributions of a sample. While the potential of SEC remains strong, our awareness of the pitfalls and challenges inherent to it and to its practice must also be ever-present. This Perspective aims to highlight some of the advantages and applications of SEC, to bring to the fore these caveats with regard to its practice, and to provide an outlook as to potential areas for expansion and growth.

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size exclusion chromatography research paper

In recent years, size exclusion chromatography (SEC) has gained valuable and impactable recognition among various chromatographic techniques. Also addressed as other names, viz. gel permeation chromatography, steric-exclusion chromatography, etc., SEC is typically taken into consideration for the fractionation and molecular weight determination of biomolecules and large macromolecules (proteins and polymers) using porous particles. A homogenous mixture of molecules dispersed in the mobile phase is introduced to the chromatographic column, which provides a solid support in the form of microscopic beads (the stationary phase). The beads act as “sieves” and purify small molecules, which become temporarily trapped inside the pores. Some of the advantages that SEC offers over other chromatographic techniques are short analysis time, no sample loss, good sensitivity, and requirement for less amount of mobile phase. In the proposed manuscript, we have deliberated various proteomic applications of size exclusion chromatography, which include the isolation of extracellular vesicles in cancer, isolation of human synovial fluid, separation of monoclonal antibodies, as well as several tandem techniques, such as deep glycoproteomic analysis using SEC-LC-MS/MS, analysis of mammalian polysomes in cells and tissues using tandem MS-SEC, SEC-SWATH-MS profiling of the proteome with a focus on complexity, etc.

Keywords: Size exclusion , chromatography , molecular weight , proteins , cancer , antibodies.

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size exclusion chromatography research paper

Title: Recent Advancements and Applications of Size Exclusion Chromatography in Modern Analysis

Volume: 19 Issue: 5

Author(s): Yogindra Kumari, Arshdeep Chopra and Rohit Bhatia*

Abstract: In recent years, size exclusion chromatography (SEC) has gained valuable and impactable recognition among various chromatographic techniques. Also addressed as other names, viz. gel permeation chromatography, steric-exclusion chromatography, etc., SEC is typically taken into consideration for the fractionation and molecular weight determination of biomolecules and large macromolecules (proteins and polymers) using porous particles. A homogenous mixture of molecules dispersed in the mobile phase is introduced to the chromatographic column, which provides a solid support in the form of microscopic beads (the stationary phase). The beads act as “sieves” and purify small molecules, which become temporarily trapped inside the pores. Some of the advantages that SEC offers over other chromatographic techniques are short analysis time, no sample loss, good sensitivity, and requirement for less amount of mobile phase. In the proposed manuscript, we have deliberated various proteomic applications of size exclusion chromatography, which include the isolation of extracellular vesicles in cancer, isolation of human synovial fluid, separation of monoclonal antibodies, as well as several tandem techniques, such as deep glycoproteomic analysis using SEC-LC-MS/MS, analysis of mammalian polysomes in cells and tissues using tandem MS-SEC, SEC-SWATH-MS profiling of the proteome with a focus on complexity, etc.

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Kumari Yogindra, Chopra Arshdeep and Bhatia Rohit*, Recent Advancements and Applications of Size Exclusion Chromatography in Modern Analysis, Current Analytical Chemistry 2023; 19 (5) . https://dx.doi.org/10.2174/1573411019666230526144816

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Chapter 5 Size-exclusion chromatography

Publisher summary.

Size-exclusion chromatography (SEC), also called “gel-permeation chromatography,” “molecular-sieve chromatography,” or “gel filtration,” separates molecules according to their sizes. Smaller molecules are retarded on a column, whereas larger ones are eluted more rapidly. As the retention time can be directly correlated with the sizes of molecules, this method is particularly useful for the determination of molecular weight. SEC is generally applicable to the separation of molecules in the range of 0.5–1000 kDa, but larger proteins or other giant molecules can also be separated. SEC can also physically separate folded macromolecules from the unfolded ones, particularly in the case of slow equilibria. The major applications of SEC include the determination of molecular weight and molecular-weight distribution of natural and synthetic polymers. Separation strongly depends on many factors such as column packing, column dimensions, flow rate, sample volume, and mobile-phase composition. Capillary SEC provides a separation strategy for microscale purification. Capillary SEC columns are best suited for direct coupling to electrospray ionization–mass spectrometry (ESI–MS) because they have comparable flow rates that can be delivered to the ESI source without splitting.

IMAGES

  1. Mechanism of size-exclusion chromatography (SEC) 83

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  2. Principle for Size-exclusion chromatography-based exosome isolation

    size exclusion chromatography research paper

  3. Figure S4. A) Size exclusion chromatography analysis of purified MtsR

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  4. Size exclusion chromatography

    size exclusion chromatography research paper

  5. Size exclusion chromatography

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  6. PPT

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  1. Part-3. Gel permeation chromatography

  2. Lecture 35: An Introduction to Chromatographic Separations(4)

  3. Size-Exclusion Chromatography (SEC) and Waters Part 1: Thinking Differently

  4. Part-2. Gel permeation chromatography

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  6. Size Exclusion Chromatography 😲 #shorts #education #viral

COMMENTS

  1. Size-Exclusion Chromatography for the Analysis of Protein Biotherapeutics and their Aggregates

    This need has renewed interest in size-exclusion chromatography (SEC). ... it was realized that SEC materials follow the same chromatographic theory as adsorption chromatography. In one of the early papers describing Sephadex, Flodin ... Pharmaceutical Research. 2010:1-14. [PMC free article] ...

  2. A brief practical review of size exclusion chromatography: Rules of

    A brief practical review of size exclusion chromatography: Rules of thumb, limitations, and troubleshooting. ... Presenting a SEC column profile in a purification paper. ... For the last 20 years it has become more and more common for research labs that perform a lot of chromatography to acquire modern chromatography systems. These systems are ...

  3. Investigate the efficacy of size exclusion chromatography for the

    Size exclusion chromatography (SEC) is a size-based separation technique, that employs a stationary phase often consisting of gel polymers through which the mobile phase transverses and gets eluted. ... Numerous research papers emphasize the benefits of integrating these two standard methods to improve both purity and yield. SEC elutes, crucial ...

  4. Size Exclusion Chromatography Method for Purification of ...

    Size exclusion high-performance liquid chromatography of small solutes in Column Handbook for Size Exclusion Chromatography (ed. Wu, C. S.) 249-266 (Academic Press Inc., 1999). Cannell, R.

  5. Size-exclusion chromatography as a useful tool for the assessment of

    At least one detector in SEC is required to detect the eluting sample. In many cases, SEC instruments for research are fitted with 3 or 4 detectors in series. ... Additional information can be found in review papers (Kilz & Pasch, 2000; Striegel et al., 2009) and in the ... Modern size-exclusion chromatography (2nd ed.) Hoboken: Wiley. 10.1002 ...

  6. Size-Exclusion Chromatography: A Twenty-First Century Perspective

    Size-Exclusion Chromatography: A Twenty-First Century Perspective 309 1 3 detector such as a dierential refractometer (DRI) or Ultra-violet/Visible spectrophotometer (UV/Vis). For example, the general concepts behind an SEC separation were explained in the second paragraph of this paper, concisely and without any equations.

  7. Size exclusion chromatography

    Abstract Size exclusion chromatography, SEC is one of the most popular methods for the separation of different kinds of macromolecules. ... Drug Development Research; Drug Testing and Analysis; Electroanalysis; Electrophoresis; ... Dúbravská cesta 9, 84236 Bratislava, Slovakia Fax:+421-2-54775923Search for more papers by this author. First ...

  8. Mathematical Modeling of Size Exclusion Chromatography

    A mathematical model of the size exclusion chromatography (SEC) process in chromatographic columns has been developed. It considers the following three mass transfer processes in the SEC column: axial dispersion in the bulk-fluid phase, interfacial film mass-transfer between the stationary and mobile phases, and diffusion of solutes within the ...

  9. Size-Exclusion Chromatography: A Twenty-First Century Perspective

    As is, perhaps, the case with many of its users, size-exclusion chromatography (SEC), born in the 1950s, has gone from being the "hot, young, new thing" in the 1960s and early-70s, to irresponsible youth in the late-70s and 1980s (when at-best-dubious calibrant-relative results ran rampant through the literature), to questioning adult in the 1990s (when the combined applications on-line ...

  10. Recent Advancements and Applications of Size Exclusion Chromatography

    In recent years, size exclusion chromatography (SEC) has gained valuable and impactable recognition among various chromatographic techniques. Also addressed as other names, viz. gel permeation chromatography, steric-exclusion chromatography, etc., SEC is typically taken into consideration for the fractionation and molecular weight determination of biomolecules and large macromolecules ...

  11. PDF Ms. Kiran R. Ghule *, Mr. Nitin Neharkar, Mrs. Bhagyashri Shelar,

    Mechanism of Size Exclusion Chromatography (1) Size exclusion also referred to as gel filtration chromatography may be a case of liquid-liquid partition chromatography, during which the solute molecules are get distributed in between two liquid phases, (i) liquid within the gel pores and (ii) liquid outside the gel. The size exclusion might be ...

  12. Chapter 5 Size-exclusion chromatography

    Chapter 5 Size-exclusion chromatography. Size-exclusion chromatography (SEC), also called "gel-permeation chromatography," "molecular-sieve chromatography," or "gel filtration," separates molecules according to their sizes. Smaller molecules are retarded on a column, whereas larger ones are eluted more rapidly.

  13. Size Exclusion Chromatography: A Teaching Aid for Physical Chemistry

    By examining the underlying concepts of size exclusion chromatography (SEC), students can gain greater appreciation of and insight into the science and utility of thermodynamics, as well as solution properties of macromolecules. In this paper we will show how the separation mechanism that governs SEC, an unusual form of chromatography, can be ...

  14. Size-Exclusion Chromatography for the Analysis of Protein

    Behavior of Insulin on High-Performance Size Exclusion Chromatography at Neutral pH. Journal of Pharmaceutical and Biomedical Analysis 2010 , 52 (2), 195-202.

  15. Protein analysis with size exclusion chromatography (SEC)

    Size exclusion chromatography (SEC) is currently the most powerful chromatography technique for obtaining reliable information about the size of biomolecules under native conditions. As such, it is widely used in several different analytical applications from basic research to quality control of biotherapeutics. This white paper presents ...

  16. PDF An Introduction to Gel Permeation Chromatography and Size Exclusion

    Gel permeation/size exclusion chromatography 5 Chapter 2 - GPC/SEC overview 6 Polymers 6 Size matters 6 How does GPC/SEC work 7 Who uses GPC/SEC, what for and why 8 ... industry for research and development of new compounds. The instruments can be complex and expensive, or simple and inexpensive, so much so that practical chromatography can ...

  17. (PDF) Size Exclusion Chromatography

    Molecular Exclusion Chromatography. Molecules are separated according to their size. SEC separation of two macromo lecular sizes: 1.Sample mixture before ente ring the column. packing. 2.Sample ...

  18. Size-exclusion chromatography

    Size-exclusion chromatography, also known as molecular sieve chromatography, is a chromatographic method in which molecules in solution are separated by their size, and in some cases molecular weight. It is usually applied to large molecules or macromolecular complexes such as proteins and industrial polymers. Typically, when an aqueous solution is used to transport the sample through the ...

  19. Size Exclusion Chromatography

    The size range of 4 to 12 µm is traditionally the standard for analytical SEC resins. However, the trend is towards smaller particles of < 2 µm, with the use of ultra high-performance liquid chromatography (UHPLC) systems for even faster separations in high-throughput mode.