# US 12,214,299 B2 · Composition, system and method for removal of detergents from aqueous solutions

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- Number: US 12,214,299 B2
- Document ID: US12214299B2
- Status: Granted
- Kind: B2
- Application number: 16/305,191
- Filed: 2017-05-30
- Published: 2020-10-08
- Granted: 2025-02-04
- Earliest claimed: 2016-05-31
- Assignees: Dna Genotek Inc.
- Applicants: Dna Genotek Inc.
- Inventors: Hyman Chaim Birnboim; Rajeev Mani Nepal; Bitapi Ray; Jessica Lynne Gage; Christopher Gordon Askew
- Web page: https://patentgrep.com/patent/US12214299B2

## CPC

- B01D 17/047: {with separation aids}
- C02F 1/26: by extraction
- B01D 17/00: Separation of liquids, not provided for elsewhere, e.g. by thermal diffusion (devices for separating or removing fatty or oily substances or similar floating material from water, waste water, or sewage C02F1/40; cleaning or keeping clear the surface of open water from oil or like materials E02B15/04; arrangements for separating lubricants from refrigerants F25B43/02)
- B01D 17/0217: {by centrifugal force}
- C02F 1/385: {by centrifuging suspensions (centrifuges B04B)}
- C02F 1/40: Devices for separating or removing fatty or oily substances or similar floating material (cleaning or keeping clear the surface of open water from oil or like materials E02B15/04; devices in sewers for separating liquid or solid substances from sewage E03F5/14, e.g. for use in drains leading to the sewer E03F5/16)
- C07C 303/44: Separation; Purification
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C02F 2101/301: {Detergents, surfactants}
- B01D: SEPARATION (separating solids from solids by wet methods B03B, B03D; by pneumatic jigs or tables B03B; by other dry methods B07; magnetic or electrostatic separation of solid materials from solid materials or fluids, separation by high-voltage electric fields B03C; centrifuges, vortex apparatus B04; presses per se for squeezing-out liquid from liquid-containing material B30B9/02; treatment of water C02F, e.g. softening by ion-exchange C02F1/42; {arrangements of air intake cleaners in gas turbine plants F02C7/05} ; arrangements or mounting of filters in air-conditioning, air-humidification or ventilation F24F13/28)
- C02F: TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE (separation in general B01D; special arrangements on waterborne vessels of installations for treating water, waste water or sewage, e.g. for producing fresh water, B63J; adding materials to water to prevent corrosion C23F; treating radioactively-contaminated liquids G21F9/04; regeneration of reactants for recirculation into processes, see the relevant places for the processes)
- C07C: ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

A system, composition, method and kit for removing a detergent, such as an anionic detergent, from an aqueous solution, comprising a salt, a water immiscible alcohol of Formula I: R1—OH (Formula I) where R1 is an optionally substituted, linear, branched or cyclic C4-C12 alkyl; and a water immiscible halocarbon, wherein said halocarbon is miscible with said alcohol of Formula I. The system can be used on aqueous solutions that contain detergents (such as Sodium Dodecyl Sulphate (SDS), for example), and any detergent-associated or detergent-bound molecules that may be present in the aqueous solution, to form an aqueous phase and a non-aqueous phase, for effectively removing the detergent and any detergent-associated or detergent-bound molecules, and sequestering them into the non-aqueous phase.

## Claims

### Claim 1

A method of removing a detergent from an aqueous solution comprising said detergent, said method comprising the step of mixing the aqueous solution with:
a. a salt;
b. a water immiscible alcohol of Formula I
R1—OH (Formula I)
where R1 is an optionally substituted, linear, branched or cyclic C4-C12 alkyl; and
c. a water immiscible halocarbon, wherein said halocarbon is miscible with said alcohol of Formula I,
to form a two-phase mixture comprising an aqueous phase and a non-aqueous phase, wherein more than 99% wt/vol of the detergent is in the non-aqueous phase; and
wherein the halocarbon is present at a concentration sufficient to increase the density of the non-aqueous phase to greater than the density of the aqueous phase, wherein the concentration of halocarbon in the alcohol is up to 80% (v/v),
wherein the ratio of the total volume of the salt plus the alcohol plus the halocarbon to the volume of the aqueous solution comprising the detergent is from 1:9 to 2:1.

### Claim 2 (depends on 1)

The method of claim 1, wherein the detergent is an anionic detergent.

### Claim 3 (depends on 1)

The method of claim 1, wherein the method further removes a detergent-associated and/or detergent-bound molecule from the aqueous solution; optionally, wherein the detergent-associated and/or detergent-bound molecule is a protein.

### Claim 4 (depends on 1)

The method of claim 1, wherein the alcohol and the halocarbon are combined prior to mixing with the aqueous solution;
optionally, wherein the method additionally comprises the step of separating the aqueous phase from the two-phase mixture;
optionally, wherein the method additionally comprises the step of centrifuging the mixture to facilitate separation of the two phases.

### Claim 5 (depends on 1)

The method of claim 1, wherein the method comprises one or more of the following characteristics:
the concentration of the salt in the aqueous phase of said two-phase mixture is from 30 mM to 0.5 M; and
the concentration of halocarbon in the alcohol is up to 50% (v/v).

### Claim 6 (depends on 1)

The method of claim 1, wherein the ratio of the total volume of the salt plus the alcohol plus the halocarbon to the volume of the aqueous solution comprising the detergent is 1:5.

### Claim 7 (depends on 1)

The method of claim 1, further comprising the step of directly using a portion of the aqueous phase from the two-phase mixture for a downstream application selected from a polymerase chain reaction (PCR), library preparation, or nucleotide sequencing; wherein the PCR is optionally RT-PCR.

### Claim 8 (depends on 1)

The method of claim 1, wherein the aqueous phase of the two-phase mixture comprises <0.01% wt/vol of the detergent.

### Claim 9 (depends on 1)

The method of claim 1, wherein the method comprises one or more of the following characteristics:
(a) the salt is a quaternary ammonium salt or an alkali metal salt; optionally, wherein the salt is ammonium acetate, ammonium chloride, ammonium sulfate, a betaine salt, choline chloride, diethylamine hydrochloride, dimethylethanolamine, ethanolamine, ethylammonium chloride, methylammonium chloride, tetra-n-butylammonium acetate, tetraethylammonium chloride monohydrate, tetramethylammonium chloride, tetra-n-propylammonium chloride, methyltri-n-butylammonium chloride, triethylamine hydrochloride, triethylmethylammonium chloride, trimethylamine hydrochloride, an acid salt of 1-butylamine, an acid salt of 1-pentylamine, an acid salt of 1-hexylamine, an acid salt of 1-dodecylamine, lithium chloride; sodium chloride, potassium chloride, or a combination thereof;
(b) the water immiscible alcohol is 1-butanol, 3-methyl-1-butanol, 2-butanol, 1-heptanol, 1,2-hexanediol, 1-hexanol, 2-nonanol, 1-pentanol, 4-methyl-2-pentanol, cyclopentanol, 1-undecanol or a combination thereof;
(c) the halocarbon is 1-bromo-3-chloropropane, 1-bromo-6-chlorohexane, bromodichloromethane (BDCM), chlorodibromomethane, chloroform, 2-iodopropane, poly(chlorotrifluoroethylene), or a combination thereof; and
(d) the detergent is sodium dodecyl sulfate (SDS).

### Claim 10 (depends on 1)

The method of claim 1, wherein the method comprises one or more of the following characteristics:
(a) the salt is ammonium chloride;
(b) the water immiscible alcohol is 1-butanol, 1-pentanol, cyclopentanol, 1-hexanol, 1-heptanol, 2-nonanol or 1-undecanol; and
(c) the halocarbon is BDCM or polychlorotrifluoroethylene.

### Claim 11 (depends on 1)

The method of claim 1, wherein the aqueous solution is a biological sample; optionally, wherein the biological sample is a modified biological sample comprising a sample storage, lysis or extraction composition.

### Claim 12 (depends on 1)

The method of claim 1, wherein the salt is ammonium chloride, the alcohol is 1-pentanol, and the halocarbon is bromodichloromethane or polychlorotrifluoroethylene.

### Claim 13 (depends on 1)

The method of claim 1, wherein the method comprises one or more of the following characteristics:
(a) a reducing agent; optionally, wherein the reducing agent is dithiothreitol (DTT), mercaptohexanol, mercaptoundecanol, dimercaptopropanol or mercaptobutanol;
(b) a chelator and/or a buffer; optionally, wherein the chelator is ethylene glycol tetraacetic acid (EGTA), (2-Hydroxyethyl)ethylenediaminetriacetic acid (HEDTA), diethylene triamine pentaacetic acid (DTPA), NitriloTriAcetic Acid (NTA), ethylenediaminetriacetic acid (EDTA), cyclohexanediaminetetraacetic acid (CDTA), citrate anhydrous, sodium citrate, calcium citrate, ammonium citrate, ammonium bicitrate, citric acid, diammonium citrate, ferric ammonium citrate, lithium citrate, or any combination thereof; and
(c) a lipophilic stain; optionally, wherein the lipophilic stain is Nile Red, 9-Anthryldiazomethane, Fluorol Yellow 088, N,N,N-Trimethyl-4-(6-phenyl-1,3,5-hexatrien-1-yl) phenylammonium p-toluenesulfonate (TMA-DPH), 3,3′-Dioctadecyloxacarbocyanine percholorate, 1,6-Diphenyl-1,3,5-hexatriene, Sudan III, Sudan Orange G, Nile Blue chloride or Solvent Blue 37.

### Claim 14 (depends on 1)

The method of claim 1, wherein the aqueous solution comprising said detergent has a concentration of detergent up to 10% (w/v),
wherein, following the step of mixing the aqueous solution with the salt, the water immiscible alcohol of Formula I, and the water immiscible halocarbon to form the two-phase mixture:
the concentration of the salt in the aqueous phase of said two-phase mixture is from 30 mM to 0.5 M, and
the aqueous phase of the two-phase mixture comprises <0.01% wt/vol of the detergent.